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

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

Cigarette smoke-induced accumulation of lung dendritic cells is interleukin-1α-dependent in mice

Abstract Background Evidence suggests that dendritic cells accumulate in the lungs of COPD patients and correlate with disease severity. We investigated the importance of IL-1R1 and its ligands IL-1α and β to dendritic cell accumulation and maturation in response to cigarette smoke exposure. Methods Mice were exposed to cigarette smoke using a whole body smoke exposure system. IL-1R1-, TLR4-, and IL-1α-deficient mice, as well as anti-IL-1α and anti-IL-1β blocking antibodies were used to study the importance of IL-1R1 and TLR4 to dendritic cell accumulation and activation. Results Acute and chronic cigarette smoke exposure led to increased frequency of lung dendritic cells. Accumulation and activation of dendritic cells was IL-1R1/IL-1α dependent, but TLR4- and IL-1β-independent. Corroborating the cellular data, expression of CCL20, a potent dendritic cells chemoattractant, was IL-1R1/IL-1α-dependent. Studies using IL-1R1 bone marrow-chimeric mice revealed the importance of IL-1R1 signaling on lung structural cells for CCL20 expression. Consistent with the importance of dendritic cells in T cell activation, we observed decreased CD4+ and CD8+ T cell activation in cigarette smoke-exposed IL-1R1-deficient mice. Conclusion Our findings convey the importance of IL-1R1/IL-1α to the recruitment and activation of dendritic cells in response to cigarette smoke exposure.</p

Akt and ubiquitin-proteasome signaling pathways.

<p>On the left side, upon stimulation of their respective receptor, insulin or insulin-growth factor 1 stimulates phosphorylation of Akt. Protein synthesis is then promoted through activation of p70S6K and inhibition of GSK-3β. On the right side, following stimulation by the appropriate stimuli (pro-inflammatory cytokines, oxidative stress, lipopolysaccharide, etc) NF-κB becomes activated and transcribes, among others, MuRF1. In pro-atrophic conditions, FoxO also transcribes MuRF1 and Atrogin-1. These E3-ligases increase total polyubiquitination and therefore promote protein degradation through the proteasome. Mitogen activated protein kinase p38 is also known to induce muscle protein degradation. In addition to promoting synthesis, activated Akt is an inhibitor of protein degradation by restraining nuclear translocation of FoxO. Activation = Filled arrows Inhibition = Hammer head lines Translocation = Dashed arrows.</p

Pro-degradation and anti-synthesis changes induced by chronic cigarette smoke exposure are reversible.

<p>BALB/c mice were either exposed to room air or cigarette smoke twice a day, five days a week, for 24 weeks. All mice were then exposed to room air for an additional 60 days, without smoke exposure. MuRF1 (A), Atrogin-1 (B) and FoxO3 (C) mRNA levels were measured by real-time PCR. Protein levels of MuRF1 (D), polyubiquitin linked to lysine 48 (E), total p38 (F), phosphorylated Akt/total Akt ratio (G) and total GSK-3β (H) were assessed by Western Blot and normalized to tubulin signal. Results are mean ± SEM of 5 mice. *p<0.05 Room air control mice = Light gray Smoke-exposed mice = Black.</p

Soleus muscle weight but not gastrocnemius is normalized following smoking cessation.

<p>BALB/c mice were either exposed to room air or cigarette smoke twice a day, five days a week, for 24 weeks. All mice were then exposed to room air for an additional 60 days, without smoke exposure. Gastrocnemius (A) and soleus (B) muscles were excised and weighted. Results are mean ± SEM of 5 mice. *p<0.05 Room air control mice = Light gray Smoke-exposed mice = Black.</p

Primer sequences used for PCR analyses.

<p>Definition of abbreviations: FoxO3 = Forkhead box-containing protein O3, IL = Interleukin, MuRF = Muscle RING finger, TNF = Tumor necrosis factor, VEGF = vascular endothelial growth factor.</p

Cigarette smoke exposure decreases whole body weight and skeletal muscle mass.

<p>BALB/c mice were either exposed to room air or cigarette smoke twice a day, five days a week, for 8 or 24 weeks. Body weight was measured prior to sacrifice (A). Tibia bones were collected from mice exposed 24 weeks to smoke or room air and measured (B). Gastrocnemius (C) and soleus (D) muscles were excised and weighted. Results are mean ± SEM of 9 or 10 mice. *p<0.05. Room air control mice = Light gray Smoke-exposed mice = Black.</p

Cigarette smoke exposure induces inflammatory, oxidative and pro-angiogenic states in the skeletal muscle that are partially reversible.

<p>BALB/c mice were either exposed to room air or cigarette smoke twice a day, five days a week, for 8 or 24 weeks (9–10 mice per group). Following this protocol, a sub-group (5 mice per group) of control mice and 24-week smoke exposed mice were exposed to room air for an additional 60 days. TNF-α (A), IL-1β (B), IL-6 (C) and VEGF (E) mRNA levels were measured by real-time PCR. Total protein oxidation (D) were assessed by Western Blot and normalized to tubulin signal. Results are mean ± SEM of 5–10 mice. *p<0.05 Room air control mice = Light gray Smoke-exposed mice = Black Ex-smoking mice = Dark gray.</p