Multi-walled carbon nanotubes (MWCNTs) are an inhomogeneous group of nanomaterials that vary in lengths, shapes and types of metal contamination, which makes hazard evaluation difficult. Here we present a toxicogenomic analysis of female C57BL/6 mouse lungs following a single intratracheal instillation of 0, 18, 54 or 162 μg/mouse of a small, curled (CNT(Small), 0.8 ± 0.1 μm in length) or large, thick MWCNT (CNT(Large), 4 ± 0.4 μm in length). The two MWCNTs were extensively characterized by SEM and TEM imaging, thermogravimetric analysis, and Brunauer-Emmett-Teller surface area analysis. Lung tissues were harvested 24h, 3 days and 28 days post-exposure. DNA microarrays were used to analyze gene expression, in parallel with analysis of bronchoalveolar lavage fluid, lung histology, DNA damage (comet assay) and the presence of reactive oxygen species (dichlorodihydrofluorescein assay), to profile and characterize related pulmonary endpoints. Overall changes in global transcription following exposure to CNT(Small) or CNT(Large) were similar. Both MWCNTs elicited strong acute phase and inflammatory responses that peaked at day 3, persisted up to 28 days, and were characterized by increased cellular influx in bronchoalveolar lavage fluid, interstitial pneumonia and gene expression changes. However, CNT(Large) elicited an earlier onset of inflammation and DNA damage, and induced more fibrosis and a unique fibrotic gene expression signature at day 28, compared to CNT(Small). The results indicate that the extent of change at the molecular level during early response phases following an acute exposure is greater in mice exposed to CNT(Large), which may eventually lead to the different responses observed at day 28.
BackgroundThe toxicity of dusts from mechanical abrasion of multi-walled carbon nanotube (CNT) epoxy nanocomposites is unknown. We compared the toxic effects of dusts generated by sanding of epoxy composites with and without CNT. The used CNT type was included for comparison.MethodsMice received a single intratracheal instillation of 18, 54 and 162 μg of CNT or 54, 162 and 486 μg of the sanding dust from epoxy composite with and without CNT. DNA damage in lung and liver, lung inflammation and liver histology were evaluated 1, 3 and 28 days after intratracheal instillation. Furthermore, the mRNA expression of interleukin 6 and heme oxygenase 1 was measured in the lungs and serum amyloid A1 in the liver. Printex 90 carbon black was included as a reference particle.ResultsPulmonary exposure to CNT and all dusts obtained by sanding epoxy composite boards resulted in recruitment of inflammatory cells into lung lumen: On day 1 after instillation these cells were primarily neutrophils but on day 3, eosinophils contributed significantly to the cell population. There were still increased numbers of neutrophils 28 days after intratracheal instillation of the highest dose of the epoxy dusts. Both CNT and epoxy dusts induced DNA damage in lung tissue up to 3 days after intratracheal instillation but not in liver tissue. There was no additive effect of adding CNT to epoxy resins for any of the pulmonary endpoints. In livers of mice instilled with CNT and epoxy dust with CNTs inflammatory and necrotic histological changes were observed, however, not in mice instilled with epoxy dust without CNT.ConclusionsPulmonary deposition of epoxy dusts with and without CNT induced inflammation and DNA damage in lung tissue. There was no additive effect of adding CNT to epoxies for any of the pulmonary endpoints. However, hepatic inflammatory and necrotic histopathological changes were seen in mice instilled with sanding dust from CNT-containing epoxy but not in mice instilled with reference epoxy.Electronic supplementary materialThe online version of this article (doi:10.1186/s12989-016-0148-2) contains supplementary material, which is available to authorized users.
The tumor suppressor VHL (von Hippel-Lindau) protein is a substrate receptor for Ubiquitin Cullin Ring Ligase complexes (CRLs), containing a BC-box domain that associates to the adaptor Elongin B/C. VHL targets hypoxia-inducible factor 1α to proteasomedependent degradation. Gam1 is an adenoviral protein, which also possesses a BC-box domain that interacts with the host Elongin B/C, thereby acting as a viral substrate receptor. Gam1 associates with both Cullin2 and Cullin5 to form CRL complexes targeting the host protein SUMO enzyme SAE1 for proteasomal degradation. We show that Gam1 protein expression induces VHL protein degradation leading to hypoxia-inducible factor 1α stabilization and induction of its downstream targets. We also characterize the CRL-dependent mechanism that drives VHL protein degradation via proteasome. Interestingly, expression of Suppressor of Cytokine Signaling (SOCS) domain-containing viral proteins and cellular BC-box proteins leads to VHL protein degradation, in a SOCS domain-containing manner. Our work underscores the exquisite ability of viral domains to uncover new regulatory mechanisms by hijacking key cellular proteins.ubiquitylation | oncoviral proteins | hypoxia U biquitylation is a posttranslational modification that involves the tagging of protein substrates with ubiquitin moieties. Ubiquitin transfer requires the coordinated and subsequent activities of the ubiquitin-activating E1 enzyme, ubiquitin-conjugating E2 enzymes, and ubiquitin E3 ligases. E3 ligases show substrate specificity and can be grouped into three families based on their different E2-docking domains. CRLs (Cullin RING Ligases) are the largest subfamily of RING domain ligases (1). They display the same modular structure: a scaffold subunit (Cullin), a RING subunit, an adaptor, and a substrate-receptor subunit to recruit the specific protein target to be ubiquitylated (2). Each Cullin associates with a specific adaptor subunit, except for Cullin2 and Cullin5, which both interact with the Elongin B and Elongin C (hereafter EloB/C) heterodimer (3). The EloB/C heterodimer is specifically bound by substrate receptors that contain a minimal consensus sequence (called BC-box motif) (4-6) usually included in the SOCS (Suppressor of Cytokine Signaling) domain of several substrate-receptor subunits (7,8). The consensus sequence and the function of the BC-box motif were first described for SOCS proteins (5, 8, 9), a family of downstream effectors of cytokine signaling cascade, involved in the negative feedback regulation of this pathway.Several viruses encode for their own substrate receptors possessing the BC-box motif and are thus able to interact with EloB/ C cellular adaptor, thereby modifying the cellular ubiquitin E3 ligase complex and their target proteins selection (10-17). We have demonstrated that the CELO (Chicken Embryo Lethal Orphan) Adenovirus early protein Gam1 is one of such BC boxcontaining proteins that is able to associate with both Cullin2 and Cullin5 to reconstitute active E3 ligase complexes and tar...
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