Understanding the Variable Drivers of Toxicity for the Broad Class of Carbon Nanotubes and Nanofibers from U.S. Facilities

Pulmonary exposure to carbon nanotubes or nanofibers (CNT/F) is known to induce inflammation, toxicity, or tumorigenesis, and is a concern in the occupational setting. U. S. facility employees are at risk of inhalation exposure of multi-walled carbon nanotubes and carbon nanofibers during primary and secondary manufacturing. To date, only one MWCNT, Mitsui-7 has been classified as possibly carcinogenic to humans (Group 2B), while all other materials were subsequently categorized as unclassifiable (Group 3). This class of material has recently been listed as a high priority to the International Agency for Research on Cancer due to this significant knowledge gap. Furthermore, expressed desire to better understand the toxicity profiles of these materials has emerged from the National Institute for Occupational Safety and Health. While human research to date is limited, the use of in in vivo and in vitro model systems can be implemented for the assessment of toxicity outcomes following respiratory exposure to CNT/F. The goal of this study was to generate an accurate an effective safety profile of MWCNT and CNFs from U. S. facilities, and to adapt a multi-disciplinary approach using machine learning to identify pertinent physicochemical characteristics that act as drivers of these toxicity outcomes. This study established toxicity profiles from male C57BL6/J mice aged 8-10 weeks exposed to either 4 or 40 µg of one of nine different CNT/F via oropharyngeal aspiration as well as human epithelial BEAS-2B cells (0-24 µg/ml), differentiated THP-1 cells (0-120 µg/ml), and human fibroblasts (0-2 µg/ml) for four primary outcomes of genotoxicity, inflammation, pathology, and translocation. The nine materials used in this study had a wide range of characteristics including diameter (6-397 nm), length (0.1-50 µm), surface area (18-238 m2/g), aspect ratio (2-1396), residual metal catalyst (0.3-6.2 %), density (0.007-0.220 g/cm3), etc., to consider. Toxicity profiles were generated regarding these four primary toxicity outcomes, and both supervised and unsupervised machine learning was used to identify the key drivers of these adverse health effects. While some physicochemical characteristics were determined to be key drivers of specific toxicity outcomes, different characteristics were essential when considering other toxicity endpoints. No single characteristic could be used as a toxicity predictor, therefore, multifactorial processes, or combination of characteristics, were necessary for an accurate and effective prediction model for responses. The study identified physicochemical drivers of CNT/F toxicity using an integrated approach, combining experimental evidence with computational modeling, with potential for broad application. This study provides necessary information for the consideration of the potential human health effects that can result from CNT/F exposure. The safety profiles and identified drivers of toxicity may be useful for future predictive risk assessment studies and translational studies as well as contributing to safety-by-design for future material designs

Nanotoxicology

Carbon nanotubes (CNT) have been developed into new materials with a variety of industrial and commercial applications. In contrast, the physicochemical properties of CNT at the nanoscale render them the potency to generate toxic effects. Indeed, the potential health impacts of CNT have drawn a great deal of attention in recent years, owing to their identified toxicological and pathological consequences including cytotoxicity, inflammation, fibrosis, genotoxicity, tumorigenesis, and immunotoxicity. Understanding the mechanisms by which CNT induce toxicity and pathology is thus urgently needed for accurate risk assessment of CNT exposure in humans, and for safe and responsible development and commercialization of nanotechnology. Here, we summarize and discuss recent advances in this area with a focus on the molecular interactions between CNT and mammalian systems, and the signaling pathways important for the development of CNT toxicity such as the NF-\uce\ubaB, NLRP3 inflammasome, TGF-\uce\ub21, MAPK, and p53 signaling cascades. With the current mechanistic evidence summarized in this review, we expect to provide new insights into CNT toxicology at the molecular level and offer new clues to the prevention of health effects resulting from CNT exposure. Moreover, we disclose questions and issues that remain in this rapidly advancing field of nanotoxicology, which would facilitate ascertaining future research directions.CC999999/Intramural CDC HHS/United States2016-01-11T00:00:00Z25676622PMC470767

A systems genetics approach to the characterization of differential low dose radiation responses in BXD recombinant inbred mice

High doses of radiation (HDR) are clearly detrimental to human health, but relatively little is known about the health consequences following exposure to low doses of radiation (LDR, \u3c10cGy). Understanding the risks associated with LDR is of great importance to the general public due to the recent dramatic increase in diagnostic radiological imaging. While HDR clearly suppress immune function, there is evidence that LDR can be immunostimulatory. Within the organism, defining the consequences of LDR is further complicated by the impact of genetic background, particularly in systems such as the immune system for which both radiosensitivity and genetic effects are profound. We addressed the issue of genetic susceptibility to LDR using the immune system as a target system and treated the LDR response as a complex trait analyzed using a systems genetics framework. Using the BXD recombinant inbred strain mouse panel as a genetic reference population allowed us to address the radiation response within the context of natural genetic variation. Our overarching hypothesis is that, within a population, the immunological effects of LDR exposure depend in part on the individual’s baseline immunoprofile and gene expression which are ultimately dependent upon genetic background. We began by establishing the immunophenotypic variation (i.e., T:B cell ratio, CD4:CD8 ratio) within the BXD panel and used baseline spleen transcriptome profiling to identify putative candidate genes controlling these traits, specifically Acp1 and Ptprk for CD4:CD8 ratio. The same set of BXD strains was exposed to LDR (10cGy gamma radiation) to determine effects on immune function and oxidative stress. LDR significantly enhanced neutrophil phagocytosis in a manner that was independent of genetic background. In contrast, genetic background significantly impacted LDR-induced changes in spleen superoxide dismutase activity. By integrating these results with our previous analyses of BXD RI strains, we have demonstrated that baseline expression of Sod2 correlates with LDR-induced SOD activity, and baseline CD4:CD8 ratio is inversely correlated with LDR-induced neutrophil phagocytosis. In addition, spleen transcriptomic data from the BXD parental strains further highlighted the impact of genetic background on LDR responses. These data provide the groundwork for predicting LDR responses using baseline expression, immunophenotypes, and/or genotype

The Effect of Physicochemical Properties on the Multiwalled Carbon Nanotube-induced Genotoxicity and Carcinogenesis

Multiwalled carbon nanotubes (MWCNT) are one of the most exciting industrial materials of our time. They are used in life-saving medical therapeutics and many commercial products that could make day-to-day life seem effortless. However, previous toxicological research has demonstrated this material to be significantly genotoxic in both in vitro and in vivo models and potentially carcinogenic in the lung. The unique physiochemical properties of MWCNT make respiratory exposures likely in workers. Combining the genotoxic effects with the potential for lung deposition in the workplace, MWCNT should be considered as a potential health hazard. Altering the physiochemical properties of MWCNT has been shown to effect toxicity, however there has been limited research on how this effects the mechanism of genotoxicity and carcinogenicity.;Therefore, the aim of the first study was to determine the effect of MWCNT diameter on the mechanism of genotoxicity. Previous research has demonstrated that exposure to MWCNT material both in vitro and in vivo induces DNA damage leading to significant aneuploidy. It is known that the microtubules that make up the mitotic spindle are 20 nm in diameter. Therefore, human lung epithelial cells were exposed to MWCNT material 10-20 nm in diameter at occupationally-relevant doses. Significant genotoxicity was observed as arrests in the G1/S phase of the cell cycle. Exposure to MWCNT led to significantly increased mitotic spindle aberrations that were predominately monopolar in morphology and fragmented centrosomes. Exposure to the highest dose produced 62% aneuploidy cells that was significantly greater than control. Aneuploidy was the result of both gains and losses of chromosomes 1 and 4.;The aim of the second study was to determine the effect of MWCNT chemical composition on the mechanism of genotoxicity. Previous research has demonstrated that eliminating metal contaminates in the MWCNT through high-temperature treatment post-synthesis (MWCNT-HT) or incorporating nitrogen into the lattice structure of the walls of MWCNT structure during synthesis (MWCNT-ND) can potentially reduce the toxicity of the pristine material (MWCNT-7). Therefore, two types of human lung epithelial cells were exposed to MWCNT-7, MWCNT-HT, and MWCNT-ND in a dose-response. Significant genotoxicity was observed in two cell types through arrests in the cell cycle that indicate centrosomal damage after exposure to each MWCNT material. Exposure to each MWCNT material also led to significantly increased mitotic spindle aberrations and fragmented centrosomes. Exposure to the highest dose of MWCNT-7, HT and ND material produced 65, 58, and 53% aneuploidy cells, respectively. Detailed chromosome analysis demonstrated significantly increased frequency of fragmented centromeres and translocations between chromosomes 1 and 4.;The aim of the third study was to determine the mechanism of carcinogenicity of inhaling MWCNT-7 material via a two-stage initiation-promotion protocol. Previous research demonstrated the MWCNT-7 material to be significantly genotoxic and the potential for MWCNT material to be a tumor promoter. At 17 months post-exposure, 23.2, 51.9, 26.5, and 90.5% of mice from the control, initiating agent only, MWCNT-7 only, or initiating agent and MWCNT-7 group, respectively, had lung tumors. The tumor multiplicity, potency, and volume in the latter group was significantly greater than control indicating that MWCNT-7 material is a strong tumor promoter. Additionally, mice that received both initiating agent and MWCNT-7 demonstrated evidence of serosal tumors morphologically consistent with sarcomatous mesotheliomas.;In conclusion, these studies indicate that MWCNT material, regardless of physicochemical modification, is significantly genotoxic by disrupting the mitotic spindle and fragmenting centrosomes leading to significant aneuploidy. The MWCNT-7 material produced the greatest amount of aneuploidy. Inhalation exposure to this material was significantly carcinogenic and shown to work through the mechanism of tumor promotion rather than initiation

In vitro studies of nanoparticle interactions within the human alveolar respiratory unit

Carbon nanotubes (CNTs) have attracted interest in many industries because of their novel and beneficial properties. Future exposure to public by inhalation is expected with the dramatic increase in manufacture and use of CNTs. Concurrently urban air pollution, including the contribution of diesel exhaust particles (DEPs), is already a heavy burden on public health on a global scale. The overarching hypothesis of the study was that the physicochemical properties of carbon-based nanoparticles would determine their bioreactivity and toxicity at the alveolar unit of the lung. As I was studying both CNTs and DEPs, I had two more specific hypotheses: 1) The cellular responses would be dependent on the source of exhaust emissions which determines the size and composition of the resultant DEPs. 2) The surface charge of multi-walled carbon nanotubes (MWCNTs) would determine the magnitude of the bioreactive responses in alveolar cells. Simple and complex in vitro models utilising the human alveolar type 1 cell line (TT1) and human primary cells: alveolar epithelial type 2 (AT2), alveolar macrophage (AM) and microvascular endothelial cells (HPMVEC) were used to test the hypotheses. The development and use of a complex in vitro cell model was a vital aspect of this study as it was imperative to understand the interaction not only between nanoparticles (NPs) and cells but also to gain an insight into the interaction between cells. Performing studies in simple and complex models comparatively allowed for greater awareness of the cell-cell interactions which inform toxicological outcomes following particle uptake including reactive oxygen species (ROS) formation and inflammation. Diesel particulate matter (PM) and carbon black induced moderate endothelial cell activation characterised by IL-6 release and a pro-thrombotic response. Recently generated automobile diesel PM and biodiesel PM were notably less reactive at the epithelium than forklift diesel PM and carbon black; however as all of the NPs were internalised by the epithelial type 1 cells and macrophages, the potential chronic and systemic effects of all of the particles should be considered. The conspicuous cytotoxicity of carbon black was likely a combination of its smaller size and surface activity. The MWCNTs were acutely reactive at the alveolar epithelium, inducing cytotoxicity, inflammation (including anti-protease production at even the lowest concentrations, 0.1 µg/ml) and ROS generation. All of the MWCNTs were internalised by the alveolar cells, by active and passive uptake mechanisms, however the positively charged CNT had distinctly more interaction with the alveolar cells and thus greater uptake, and even translocation in the epithelial type 1 cells. Of the MWCNTs, MAA and APTAC induced the smallest and greatest bioreactive responses respectively, in the cellular (simple and complex) models utilised, confirming the hypothesis. The association between nanoparticle exposure in humans to a number of adverse respiratory outcomes is well-established however the mechanisms by which these objects influence human health are still only partially understood. My study demonstrated that nanoparticle toxicity is dependent on their physicochemical properties, specifically charge in the case of MWCNTs, and generally size, as well as the cell models used. Understanding the toxicity of carbon nanomaterials, both anthropogenic and engineered, which are major contaminants of our environment and determinants of cardiopulmonary toxicity, is essential to generating safer nanomaterials and limiting airborne exposure to protect human health.Open Acces

J Toxicol Environ Health B Crit Rev

Carbon nanotubes (CNT) and nanofibers (CNF) are used increasingly in a broad array of commercial products. Given current understandings, the most significant life-cycle exposures to CNT/CNF occur from inhalation when they become airborne at different stages of their life cycle, including workplace, use, and disposal. Increasing awareness of the importance of physicochemical properties as determinants of toxicity of CNT/CNF and existing difficulties in interpreting results of mostly acute rodent inhalation studies to date necessitate a reexamination of standardized inhalation testing guidelines. The current literature on pulmonary exposure to CNT/CNF and associated effects is summarized; recommendations and conclusions are provided that address test guideline modifications for rodent inhalation studies that will improve dosimetric extrapolation modeling for hazard and risk characterization based on the analysis of exposure-dose-response relationships. Several physicochemical parameters for CNT/CNF, including shape, state of agglomeration/aggregation, surface properties, impurities, and density, influence toxicity. This requires an evaluation of the correlation between structure and pulmonary responses. Inhalation, using whole-body exposures of rodents, is recommended for acute to chronic pulmonary exposure studies. Dry powder generator methods for producing CNT/CNF aerosols are preferred, and specific instrumentation to measure mass, particle size and number distribution, and morphology in the exposure chambers are identified. Methods are discussed for establishing experimental exposure concentrations that correlate with realistic human exposures, such that unrealistically high experimental concentrations need to be identified that induce effects under mechanisms that are not relevant for workplace exposures. Recommendations for anchoring data to results seen for positive and negative benchmark materials are included, as well as periods for postexposure observation. A minimum data set of specific bronchoalveolar lavage parameters is recommended. Retained lung burden data need to be gathered such that exposure-dose-response correlations may be analyzed and potency comparisons between materials and mammalian species are obtained considering dose metric parameters for interpretation of results. Finally, a list of research needs is presented to fill data gaps for further improving design, analysis, and interpretation and extrapolation of results of rodent inhalation studies to refine meaningful risk assessments for humans.20152016-01-09T00:00:00ZCC999999/Intramural CDC HHS/United StatesP30 ES001247/ES/NIEHS NIH HHS/United StatesP30 ESO1247/PHS HHS/United States26361791PMC470675

Crit Rev Toxicol

In an evaluation of carbon nanotubes (CNTs) for the IARC Monograph 111, the Mechanisms Subgroup was tasked with assessing the strength of evidence on the potential carcinogenicity of CNTs in humans. The mechanistic evidence was considered to be not strong enough to alter the evaluations based on the animal data. In this paper, we provide an extended, in-depth examination of the in vivo and in vitro experimental studies according to current hypotheses on the carcinogenicity of inhaled particles and fibers. We cite additional studies of CNTs that were not available at the time of the IARC meeting in October 2014, and extend our evaluation to include carbon nanofibers (CNFs). Finally, we identify key data gaps and suggest research needs to reduce uncertainty. The focus of this review is on the cancer risk to workers exposed to airborne CNT or CNF during the production and use of these materials. The findings of this review, in general, affirm those of the original evaluation on the inadequate or limited evidence of carcinogenicity for most types of CNTs and CNFs at this time, and possible carcinogenicity of one type of CNT (MWCNT-7). The key evidence gaps to be filled by research include: investigation of possible associations between in vitro and early-stage in vivo events that may be predictive of lung cancer or mesothelioma, and systematic analysis of dose-response relationships across materials, including evaluation of the influence of physico-chemical properties and experimental factors on the observation of nonmalignant and malignant endpoints.CC999999/Intramural CDC HHS/United StatesP42 ES013660/ES/NIEHS NIH HHS/United States2018-01-01T00:00:00Z27537422PMC555564

Antioxidant and DPPH-Scavenging Activities of Compounds and Ethanolic Extract of the Leaf and Twigs of Caesalpinia bonduc L. Roxb.

Antioxidant effects of ethanolic extract of Caesalpinia bonduc and its isolated bioactive compounds were evaluated in vitro. The compounds included two new cassanediterpenes, 1α,7α-diacetoxy-5α,6β-dihydroxyl-cass-14(15)-epoxy-16,12-olide (1)and 12α-ethoxyl-1α,14β-diacetoxy-2α,5α-dihydroxyl cass-13(15)-en-16,12-olide(2); and others, bonducellin (3), 7,4’-dihydroxy-3,11-dehydrohomoisoflavanone (4), daucosterol (5), luteolin (6), quercetin-3-methyl ether (7) and kaempferol-3-O-α-L-rhamnopyranosyl-(1Ç2)-β-D-xylopyranoside (8). The antioxidant properties of the extract and compounds were assessed by the measurement of the total phenolic content, ascorbic acid content, total antioxidant capacity and 1-1-diphenyl-2-picryl hydrazyl (DPPH) and hydrogen peroxide radicals scavenging activities.Compounds 3, 6, 7 and ethanolic extract had DPPH scavenging activities with IC50 values of 186, 75, 17 and 102 μg/ml respectively when compared to vitamin C with 15 μg/ml. On the other hand, no significant results were obtained for hydrogen peroxide radical. In addition, compound 7 has the highest phenolic content of 0.81±0.01 mg/ml of gallic acid equivalent while compound 8 showed the highest total antioxidant capacity with 254.31±3.54 and 199.82±2.78 μg/ml gallic and ascorbic acid equivalent respectively. Compound 4 and ethanolic extract showed a high ascorbic acid content of 2.26±0.01 and 6.78±0.03 mg/ml respectively.The results obtained showed the antioxidant activity of the ethanolic extract of C. bonduc and deduced that this activity was mediated by its isolated bioactive compounds

Neurotoxicity

Neurotoxins are natural or chemical compounds that can disturb the neurological system of mammals. The neurotoxic potential of a neurotoxin is a consequence that may occur if tissue concentrations surpass a particular threshold. Chemicals disrupt adult brain function by interfering with the structure and function of various neuronal pathways, circuits, and systems in diverse ways. Neurotoxicity - New Advances provides updated information about neurotoxicity and neurotoxic chemicals including nanomaterials and pesticides. It also discusses prevention and treatment strategies. This book is an instructive and valuable guide to understanding neurotoxicity and identifying neurotoxicity mechanisms and neurotoxic disorders