Genomic and proteomic profiling of responses to toxic metals in human lung cells.

Examining global effects of toxic metals on gene expression can be useful for elucidating patterns of biological response, discovering underlying mechanisms of toxicity, and identifying candidate metal-specific genetic markers of exposure and response. Using a 1,200 gene nylon array, we examined changes in gene expression following low-dose, acute exposures of cadmium, chromium, arsenic, nickel, or mitomycin C (MMC) in BEAS-2B human bronchial epithelial cells. Total RNA was isolated from cells exposed to 3 M Cd(II) (as cadmium chloride), 10 M Cr(VI) (as sodium dichromate), 3 g/cm2 Ni(II) (as nickel subsulfide), 5 M or 50 M As(III) (as sodium arsenite), or 1 M MMC for 4 hr. Expression changes were verified at the protein level for several genes. Only a small subset of genes was differentially expressed in response to each agent: Cd, Cr, Ni, As (5 M), As (50 M), and MMC each differentially altered the expression of 25, 44, 31, 110, 65, and 16 individual genes, respectively. Few genes were commonly expressed among the various treatments. Only one gene was altered in response to all four metals (hsp90), and no gene overlapped among all five treatments. We also compared low-dose (5 M, noncytotoxic) and high-dose (50 M, cytotoxic) arsenic treatments, which surprisingly, affected expression of almost completely nonoverlapping subsets of genes, suggesting a threshold switch from a survival-based biological response at low doses to a death response at high doses

Increased focal adhesion kinase- and urokinase-type plasminogen activator receptor-associated cell signaling in endothelial cells exposed to asbestos.

Exposure of low-passage endothelial cells in culture to nonlethal amounts of asbestos, but not refractory ceramic fiber-1, increases cell motility and gene expression. These changes may be initiated by the fibers mimicking matrix proteins as ligands for receptors on the cell surface. In the present study, 1- to 3-hr exposures of endothelial cells to 5 mg/cm2 of chrysotile asbestos caused marked cell elongation and motility. However, little morphological change was seen when chrysotile was added to cells pretreated with either mannosamine to prevent assembly of glycophosphatidylinositol (GPI)-anchored receptors or with herbimycin A to inhibit tyrosine kinase activity. Affinity purification of GPI-anchored urokinase-type plasminogen activator receptor (uPAR) from chrysotile-exposed cells demonstrated that asbestos altered the profile of proteins and phosphoproteins complexed with this receptor. Tyrosine kinase activities in the complexes were also increased by asbestos. Immunoprecipitations with selective monoclonal antibodies demonstrated that both chrysotile and crocidolite asbestos increase kinase activities associated with p60 Src or p120 focal adhesion kinase (FAK). Further, chrysotile also changed the profile of proteins and phosphoproteins associated with FAK in intact cells. These data suggest that asbestos initiates endothelial cell phenotypic change through interactions with uPAR-containing complexes and that this change is mediated through tyrosine kinase cascades

Cell signaling pathways elicited by asbestos.

In recent years, it has become apparent that minerals can trigger alterations in gene expression by initiating signaling events upstream of gene transactivation. These cascades may be initiated at the cell surface after interaction of minerals with the plasma membrane either through receptorlike mechanisms or integrins. Alternatively, signaling pathways may be stimulated by active oxygen species generated both during phagocytosis of minerals and by redox reactions on the mineral surface. At least two signaling cascades linked to activation of transcription factors, i.e., DNA-binding proteins involved in modulating gene expression and DNA replication, are stimulated after exposure of lung cells to asbestos fibers in vitro. These include nuclear factor kappa B (NF kappa B) and the mitogen-activated protein kinase (MAPK) cascade important in regulation of the transcription factor, activator protein-1 (AP-1). Both NF kappa B and AP-1 bind to specific DNA sequences within the regulatory or promoter regions of genes that are critical to cell proliferation and inflammation. Unraveling the cell signaling cascades initiated by mineral dusts and pharmacologic inhibition of these events may be important for the control and treatment of mineral-associated occupational diseases

Cathode & Electromagnet Qualification Status and Power Processing Unit Development Update for the Ascendant Sub-kW Transcelestial Electric Propulsion System

A review of the component-level flight qualification efforts and power processing unit development status of the Ascendant Sub-kW Transcelestial Electric Propulsion System (ASTRAEUS) program is presented. Component-level qualification efforts were undertaken for the system’s ultra-compact heaterless LaB6 hollow cathode and electromagnets, both of which employ designs bespoke to ASTRAEUS, as they represent the highest failure risks for the thruster. Through parallel long-duration wear and ignition tests, the ASTRAEUS cathode demonstrated invariant discharge performance over more than 5000 h of operation at its maximum operating current of 4 A and demonstrated more than 25,000 ignition cycles. The ASTRAEUS electromagnets completed their environmental qualification through a demonstration of more than 1200 deep thermal cycles with no indication of coil degradation (the test articles previously completed qualification-level vibration and shock testing). ASTRAEUS’s prototype power processing unit has demonstrated more than 92% total power conversion efficiency and class-leading power density & specific power density of 4.5 W/cm3 & 1670 W/kg, respectively. The various power converters found in the ASTRAEUS power processing unit are reviewed with a focus on the methods by which such high performance was achieved

Arsenic stimulates sinusoidal endothelial cell capillarization and vessel remodeling in mouse liver

Trivalent arsenic [As(III)] is a well-known environmental toxicant that causes a wide range of organ-specific diseases and cancers. In the human liver, As(III) promotes vascular remodeling, portal fibrosis, and hypertension, but the pathogenesis of these As(III)-induced vascular changes is unknown. To investigate the hypothesis that As(III) targets the hepatic endothelium to initiate pathogenic change, mice were exposed to 0 or 250 parts per billion (ppb) of As(III) in their drinking water for 5 weeks. Arsenic(III) exposure did not affect the overall health of the animals, the general structure of the liver, or hepatocyte morphology. There was no change in the total tissue arsenic levels, indicating that arsenic does not accumulate in the liver at this level of exposure. However, there was significant vascular remodeling with increased sinusoidal endothelial cell (SEC) capillarization, vascularization of the peribiliary vascular plexus (PBVP), and constriction of hepatic arterioles in As(III)-exposed mice. In addition to ultrastructural demonstration of SEC defenestration and capillarization, quantitative immunofluorescence analysis revealed increased sinusoidal PECAM-1 and laminin-1 protein expression, suggesting gain of adherens junctions and a basement membrane. Conversion of SECs to a capillarized, dedifferentiated endothelium was confirmed at the cellular level with demonstration of increased caveolin-1 expression and SEC caveolae, as well as increased membrane-bound Rac1-GTPase

The NRF2-mediated oxidative stress response pathway is associated with tumor cell resistance to arsenic trioxide across the NCI-60 panel

<p>Abstract</p> <p>Background</p> <p>Drinking water contaminated with inorganic arsenic is associated with increased risk for different types of cancer. Paradoxically, arsenic trioxide can also be used to induce remission in patients with acute promyelocytic leukemia (APL) with a success rate of approximately 80%. A comprehensive study examining the mechanisms and potential signaling pathways contributing to the anti-tumor properties of arsenic trioxide has not been carried out.</p> <p>Methods</p> <p>Here we applied a systems biology approach to identify gene biomarkers that underlie tumor cell responses to arsenic-induced cytotoxicity. The baseline gene expression levels of 14,500 well characterized human genes were associated with the GI₅₀ data of the NCI-60 tumor cell line panel from the developmental therapeutics program (DTP) database. Selected biomarkers were tested <it>in vitro</it> for the ability to influence tumor susceptibility to arsenic trioxide.</p> <p>Results</p> <p>A significant association was found between the baseline expression levels of 209 human genes and the sensitivity of the tumor cell line panel upon exposure to arsenic trioxide. These genes were overlayed onto protein-protein network maps to identify transcriptional networks that modulate tumor cell responses to arsenic trioxide. The analysis revealed a significant enrichment for the oxidative stress response pathway mediated by nuclear factor erythroid 2-related factor 2 (NRF2) with high expression in arsenic resistant tumor cell lines. The role of the NRF2 pathway in protecting cells against arsenic-induced cell killing was validated in tumor cells using shRNA-mediated knock-down.</p> <p>Conclusions</p> <p>In this study, we show that the expression level of genes in the NRF2 pathway serve as potential gene biomarkers of tumor cell responses to arsenic trioxide. Importantly, we demonstrate that tumor cells that are deficient for NRF2 display increased sensitivity to arsenic trioxide. The results of our study will be useful in understanding the mechanism of arsenic-induced cytotoxicity in cells, as well as the increased applicability of arsenic trioxide as a chemotherapeutic agent in cancer treatment.</p

Histone deacetylase inhibitors suppress mechanical stress-induced expression of RUNX-2 and ADAMTS-5 through the inhibition of the MAPK signaling pathway in cultured human chondrocytes

Objective: To investigate the inhibitory effects and the regulatory mechanisms of histone deacetylase (HDAC) inhibitors on mechanical stress-induced gene expression of runt-related transcription factor (RUNX)-2 and a disintegrin and metalloproteinase with thrombospondin motif (ADAMTS)-5 in human chondrocytes. Methods: Human chondrocytes were seeded in stretch chambers at a concentration of 5 x 10(4) cells/chamber. Cells were pre-incubated with or without HDAC inhibitors (MS-275 or trichostatin A; TSA) for 12 h, followed by uniaxial cyclic tensile strain (CTS) (0.5 Hz, 10% elongation), which was applied for 30 min using the ST-140-10 system (STREX, Osaka, Japan). Total RNA was extracted and the expression of RUNX-2, ADAMTS-5, matrix metalloproteinase (MMP)-3, and MMP-13 at the mRNA and protein levels were examined by real-time polymerase chain reaction (PCR) and immunocytochemistry, respectively. The activation of diverse mitogen-activated protein kinase (MAPK) pathways with or without HDAC inhibitors during CTS was examined by western blotting. Results: HDAC inhibitors (TSA: 10 nM, MS-275: 100 nM) suppressed CTS-induced expression of RUNX-2, ADAMTS-5, and MMP-3 at both the mRNA and protein levels within 1 h. CTS-induced activation of p38 MAPK (p38), extracellular signal-regulated kinase (ERK), and c-Jun N-terminal kinase (INK) MAPKs was downregulated by both HDAC inhibitors. Conclusion: The CTS-induced expression of RUNX-2 and ADAMTS-5 was suppressed by HDAC inhibitors via the inhibition of the MAPK pathway activation in human chondrocytes. The results of the current study suggested a novel therapeutic role for HDAC inhibitors against degenerative joint disease such as osteoarthritis

Millicurrent stimulation of human articular chondrocytes cultivated in a collagen type-I gel and of human osteochondral explants

<p>Abstract</p> <p>Background</p> <p>Here we investigate the effect of millicurrent treatment on human chondrocytes cultivated in a collagen gel matrix and on human osteochondral explants.</p> <p>Methods</p> <p>Human chondrocytes from osteoarthritic knee joints were enzymatically released and transferred into a collagen type-I gel. Osteochondral explants and cell-seeded gel samples were cultivated in-vitro for three weeks. Samples of the verum groups were stimulated every two days by millicurrent treatment (3 mA, sinusoidal signal of 312 Hz amplitude modulated by two super-imposed signals of 0.28 Hz), while control samples remained unaffected. After recovery, collagen type-I, type-II, aggrecan, interleukin-1β, IL-6, TNFα and MMP13 were examined by immunohistochemistry and by real time PCR.</p> <p>Results</p> <p>With regard to the immunostainings 3 D gel samples and osteochondral explants did not show any differences between treatment and control group. The expression of all investigated genes of the 3 D gel samples was elevated following millicurrent treatment. While osteochondral explant gene expression of col-I, col-II and Il-1β was nearly unaffected, aggrecan gene expression was elevated. Following millicurrent treatment, IL-6, TNFα, and MMP13 gene expression decreased. In general, the standard deviations of the gene expression data were high, resulting in rarely significant results.</p> <p>Conclusions</p> <p>We conclude that millicurrent stimulation of human osteoarthritic chondrocytes cultivated in a 3 D collagen gel and of osteochondral explants directly influences cell metabolism.</p