Meta-analysis of transcriptomic responses as a means to identify pulmonary disease outcomes for engineered nanomaterials

BACKGROUND: The increasing use of engineered nanomaterials (ENMs) of varying physical and chemical characteristics poses a great challenge for screening and assessing the potential pathology induced by these materials, necessitating novel toxicological approaches. Toxicogenomics measures changes in mRNA levels in cells and tissues following exposure to toxic substances. The resulting information on altered gene expression profiles, associated pathways, and the doses at which these changes occur, are used to identify the underlying mechanisms of toxicity and to predict disease outcomes. We evaluated the applicability of toxicogenomics data in identifying potential lung-specific (genomic datasets are currently available from experiments where mice have been exposed to various ENMs through this common route of exposure) disease outcomes following exposure to ENMs. METHODS: Seven toxicogenomics studies describing mouse pulmonary responses over time following intra-tracheal exposure to increasing doses of carbon nanotubes (CNTs), carbon black, and titanium dioxide (TiO(2)) nanoparticles of varying properties were examined to understand underlying mechanisms of toxicity. mRNA profiles from these studies were compared to the publicly available datasets of 15 other mouse models of lung injury/diseases induced by various agents including bleomycin, ovalbumin, TNFα, lipopolysaccharide, bacterial infection, and welding fumes to delineate the implications of ENM-perturbed biological processes to disease pathogenesis in lungs. RESULTS: The meta-analysis revealed two distinct clusters—one driven by TiO(2) and the other by CNTs. Unsupervised clustering of the genes showing significant expression changes revealed that CNT response clustered with bleomycin injury and bacterial infection models, both of which are known to induce lung fibrosis, in a post-exposure-time dependent manner, irrespective of the CNT’s physical-chemical properties. TiO(2) samples clustered separately from CNTs and disease models. CONCLUSIONS: These results indicate that in the absence of apical toxicity data, a tiered strategy beginning with short term, in vivo tissue transcriptomics profiling can effectively and efficiently screen new ENMs that have a higher probability of inducing pulmonary pathogenesis. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/s12989-016-0137-5) contains supplementary material, which is available to authorized users

Nano-risk Science: application of toxicogenomics in an adverse outcome pathway framework for risk assessment of multi-walled carbon nanotubes

BACKGROUND: A diverse class of engineered nanomaterials (ENMs) exhibiting a wide array of physical-chemical properties that are associated with toxicological effects in experimental animals is in commercial use. However, an integrated framework for human health risk assessment (HHRA) of ENMs has yet to be established. Rodent 2-year cancer bioassays, clinical chemistry, and histopathological endpoints are still considered the ‘gold standard’ for detecting substance-induced toxicity in animal models. However, the use of data derived from alternative toxicological tools, such as genome-wide expression profiling and in vitro high-throughput assays, are gaining acceptance by the regulatory community for hazard identification and for understanding the underlying mode-of-action. Here, we conducted a case study to evaluate the application of global gene expression data in deriving pathway-based points of departure (PODs) for multi-walled carbon nanotube (MWCNT)-induced lung fibrosis, a non-cancer endpoint of regulatory importance. METHODS: Gene expression profiles from the lungs of mice exposed to three individual MWCNTs with different physical-chemical properties were used within the framework of an adverse outcome pathway (AOP) for lung fibrosis to identify key biological events linking MWCNT exposure to lung fibrosis. Significantly perturbed pathways were categorized along the key events described in the AOP. Benchmark doses (BMDs) were calculated for each perturbed pathway and were used to derive transcriptional BMDs for each MWCNT. RESULTS: Similar biological pathways were perturbed by the different MWCNT types across the doses and post-exposure time points studied. The pathway BMD values showed a time-dependent trend, with lower BMDs for pathways perturbed at the earlier post-exposure time points (24 h, 3d). The transcriptional BMDs were compared to the apical BMDs derived by the National Institute for Occupational Safety and Health (NIOSH) using alveolar septal thickness and fibrotic lesions endpoints. We found that regardless of the type of MWCNT, the BMD values for pathways associated with fibrosis were 14.0–30.4 μg/mouse, which are comparable to the BMDs derived by NIOSH for MWCNT-induced lung fibrotic lesions (21.0–27.1 μg/mouse). CONCLUSIONS: The results demonstrate that transcriptomic data can be used to as an effective mechanism-based method to derive acceptable levels of exposure to nanomaterials in product development when epidemiological data are unavailable. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/s12989-016-0125-9) contains supplementary material, which is available to authorized users

Stat-6 signaling pathway and not Interleukin-1 mediates multi-walled carbon nanotube-induced lung fibrosis in mice: insights from an adverse outcome pathway framework

Abstract Background The accumulation of MWCNTs in the lung environment leads to inflammation and the development of disease similar to pulmonary fibrosis in rodents. Adverse Outcome Pathways (AOPs) are a framework for defining and organizing the key events that comprise the biological changes leading to undesirable events. A putative AOP has been developed describing MWCNT-induced pulmonary fibrosis; inflammation and the subsequent healing response induced by inflammatory mechanisms have been implicated in disease progression. The objective of the present study was to address a key data gap in this AOP: empirical data supporting the essentiality of pulmonary inflammation as a key event prior to fibrosis. Specifically, Interleukin-1 Receptor1 (IL-1R1) and Signal Transducer and Activator of Transcription 6 (STAT6) knock-out (KO) mice were employed to target inflammation and the subsequent healing response using MWCNTs as a model pro-fibrotic stressor to determine whether this altered the development of fibrosis. Results Wild type (WT) C57BL/6, IL-1R1 (KO) or STAT6 KO mice were exposed to a high dose of Mitsui-7 MWCNT by intratracheal administration. Inflammation was assessed 24 h and 28 days post MWCNT administration, and fibrotic lesion development was assessed 28 days post MWCNT administration. MWCNT-induced acute inflammation was suppressed in IL-1R1 KO mice at the 24 h time point relative to WT mice, but this suppression was not observed 28 days post exposure, and IL-1R1 KO did not alter fibrotic disease development. In contrast, STAT6 KO mice exhibited suppressed acute inflammation and attenuated fibrotic disease in response to MWCNT administration compared to STAT6 WT mice. Whole genome analysis of all post-exposure time points identified a subset of differentially expressed genes associated with fibrosis in both KO mice compared to WT mice. Conclusion The findings support the essentiality of STAT6-mediated signaling in the development of MWCNT-induced fibrotic disease. The IL-1R1 KO results also highlight the nature of the inflammatory response associated with MWCNT exposure, and indicate a system with multiple redundancies. These data add to the evidence supporting an existing AOP, and will be useful in designing screening strategies that could be used by regulatory agencies to distinguish between MWCNTs of varying toxicity

Alterations in skeletal muscle cell homeostasis in a mouse model of cigarette smoke exposure.

Skeletal muscle dysfunction is common in chronic obstructive pulmonary disease (COPD), a disease mainly caused by chronic cigarette use. An important proportion of patients with COPD have decreased muscle mass, suggesting that chronic cigarette smoke exposure may interfere with skeletal muscle cellular equilibrium. Therefore, the main objective of this study was to investigate the kinetic of the effects that cigarette smoke exposure has on skeletal muscle cell signaling involved in protein homeostasis and to assess the reversibility of these effects.A mouse model of cigarette smoke exposure was used to assess skeletal muscle changes. BALB/c mice were exposed to cigarette smoke or room air for 8 weeks, 24 weeks or 24 weeks followed by 60 days of cessation. The gastrocnemius and soleus muscles were collected and the activation state of key mediators involved in protein synthesis and degradation was assessed.Gastrocnemius and soleus were smaller in mice exposed to cigarette smoke for 8 and 24 weeks compared to room air exposed animals. Pro-degradation proteins were induced at the mRNA level after 8 and 24 weeks. Twenty-four weeks of cigarette smoke exposure induced pro-degradation proteins and reduced Akt phosphorylation and glycogen synthase kinase-3β quantity. A 60-day smoking cessation period reversed the cell signaling alterations induced by cigarette smoke exposure.Repeated cigarette smoke exposure induces reversible muscle signaling alterations that are dependent on the duration of the cigarette smoke exposure. These results highlights a beneficial aspect associated with smoking cessation

Additional file 1: Table S1. of Meta-analysis of transcriptomic responses as a means to identify pulmonary disease outcomes for engineered nanomaterials

A list of 2334 differentially expressed genes that were consistent to all of the microarray platforms employed in the studies included in the meta-analysis. (PPTX 123 kb

Additional file 1: Table S1. of Nano-risk Science: application of toxicogenomics in an adverse outcome pathway framework for risk assessment of multi-walled carbon nanotubes

Particle physicochemical properties and exposure information. (XLSX 10 kb

Additional file 3: Figure S1. of Nano-risk Science: application of toxicogenomics in an adverse outcome pathway framework for risk assessment of multi-walled carbon nanotubes

Heatmap showing the dose at which each pathway was significantly perturbed. Each column represents a post-exposure time point for the denoted MWCNT, and each row represents a signifncatly perturbed pathway. All colored cells represent the lowest dose at which the pathway is perturbed. Blank cells represent pathways that were not significantly perturbed. (PPTX 76 kb

Additional file 2: Table S2. of Nano-risk Science: application of toxicogenomics in an adverse outcome pathway framework for risk assessment of multi-walled carbon nanotubes

List of all significant pathways associated with the molecular initiating event (MIE), each key event (KE), and each associative event (AE) including the benchmark dose (BMD) value (BMDL values included in parentheses) for each MWCNT investigated at each time-point. n.a. indicates the BMD(L) could not be calculated due to poor model fit. (XLSX 18 kb