Safety Evaluation of a Prototypical Diazirine-Based Covalent Crosslinker and Molecular Adhesive.

bis-Diazirine reagents are increasingly being used as polymer crosslinkers, adhesives, and photopatterning agents in the materials sciences literature, but little effort has been made thus far to document their chemical safety profile. Here, we describe the results of a detailed toxicity assessment of a representative bis-diazirine. Safety was evaluated by a series of in vitro assays, which found the product to be non-mutagenic in bacterial tester strains TA98 and TA100, non-corrosive and non-irritating to skin, and requiring no classification for eye irritation or serious damage. While in vitro tests do not capture the integrated whole animal system, and thus cannot completely rule out the possibility of adverse responses, the results of this study suggest a desirable safety profile for bis-diazirine reagents and provide a solid foundation upon which to add in vivo assessment of safety risk and dose-response studies.

Assessment of the skin sensitization potential of fragrance ingredients using the U-SENS™ assay.

The U-SENS™ assay was developed to address the third key event of the skin sensitization adverse outcome pathway (AOP) and is described in OECD test guideline 442E, Annex II. A dataset of 68 fragrance ingredients comprised of 7 non-sensitizers and 61 sensitizers was tested in the U-SENS™ assay. The potential for fragrance ingredients to activate dendritic cells, measured by U-SENS™, was compared to the sensitization potential determined by weight of evidence (WoE) from historical data. Of the non-sensitizers, 4 induced CD86 cell surface marker ≥1.5-fold while 3 did not. Of the sensitizers, 50 were predicted to be positive in U-SENS™, while the remaining 11 were negative. Positive and negative predictive values (PPV and NPV) of U-SENS™ were 93% and 21%, respectively. No specific chemical property evaluated could account for misclassified ingredients. Assessment of parent and metabolite protein binding alerts in silico suggests that parent chemical metabolism may play a role in CD86 activation in U-SENS™. Combining the U-SENS™ assay in a "2 out of 3" defined approach with the direct peptide reactivity assay (DPRA) and KeratinoSens™ predicted sensitization hazard with PPV and NPV of 97% and 24%, respectively. Combining complementary in silico and in vitro methods to the U-SENS™ assay should be integrated to define the hazard classification of fragrance ingredients, since a single NAM cannot replace animal-based methods.

Involvement of c-Jun N-Terminal Kinase in TNF-α-Driven Remodeling.

Lung tissue remodeling in chronic obstructive pulmonary disease (COPD) is characterized by airway wall thickening and/or emphysema. Although the bronchial and alveolar compartments are functionally independent entities, we recently showed comparable alterations in matrix composition comprised of decreased elastin content and increased collagen and hyaluronan contents of alveolar and small airway walls. Out of several animal models tested, surfactant protein C (SPC)-TNF-α mice showed remodeling in alveolar and airway walls similar to what we observed in patients with COPD. Epithelial cells are able to undergo a phenotypic shift, gaining mesenchymal properties, a process in which c-Jun N-terminal kinase (JNK) signaling is involved. Therefore, we hypothesized that TNF-α induces JNK-dependent epithelial plasticity, which contributes to lung matrix remodeling. To this end, the ability of TNF-α to induce a phenotypic shift was assessed in A549, BEAS2B, and primary bronchial epithelial cells, and phenotypic markers were studied in SPC-TNF-α mice. Phenotypic markers of mesenchymal cells were elevated both in vitro and in vivo, as shown by the expression of vimentin, plasminogen activator inhibitor-1, collagen, and matrix metalloproteinases. Concurrently, the expression of the epithelial markers, E-cadherin and keratin 7 and 18, was attenuated. A pharmacological inhibitor of JNK attenuated this phenotypic shift in vitro, demonstrating involvement of JNK signaling in this process. Interestingly, activation of JNK signaling was also clearly present in lungs of SPC-TNF-α mice and patients with COPD. Together, these data show a role for TNF-α in the induction of a phenotypic shift in vitro, resulting in increased collagen production and the expression of elastin-degrading matrix metalloproteinases, and provide evidence for involvement of the TNF-α-JNK axis in extracellular matrix remodeling.

Safety evaluation of rebaudioside A produced by fermentation.

The safety of rebaudioside A, produced fermentatively by Yarrowia lipolytica encoding the Stevia rebaudiana metabolic pathway (fermentative Reb A), is based on several elements: first, the safety of steviol glycosides has been extensively evaluated and an acceptable daily intake has been defined; second, the use of Y. lipolytica, an avirulent yeast naturally found in foods and used for multiple applications; and third the high purity of fermentative Reb A and its compliance with internationally defined specifications. A bacterial reverse mutation assay and an in vitro micronucleus test conducted with fermentative Reb A provide evidence for its absence of mutagenicity, clastogenicity and aneugenicity. The oral administration of fermentative Reb A to Sprague-Dawley rats for at least 91 days did not lead to any adverse effects at consumption levels up to 2057 mg/kg bw/day for males and 2023 mg/kg bw/day for females, which were concluded to be the No Observed Adverse Effect Levels. The results were consistent with outcomes of previous studies conducted with plant-derived rebaudioside A, suggesting similar safety profiles for fermentative and plant-derived rebaudioside A. The results of the toxicity studies reported here support the safety of rebaudioside A produced fermentatively from Y. lipolytica, as a general purpose sweetener.

Silica-induced NLRP3 inflammasome activation in vitro and in rat lungs.

RATIONALE: Mineral particles in the lung cause inflammation and silicosis. In myeloid and bronchial epithelial cells the inflammasome plays a role in responses to crystalline silica. Thioredoxin (TRX) and its inhibitory protein TRX-interacting protein link oxidative stress with inflammasome activation. We investigated inflammasome activation by crystalline silica polymorphs and modulation by TRX in vitro, as well as its localization and the importance of silica surface reactivity in rats. METHODS: We exposed bronchial epithelial cells and differentiated macrophages to silica polymorphs quartz and cristobalite and measured caspase-1 activity as well as the release of IL-1ß, bFGF and HMGB1; including after TRX overexpression or treatment with recombinant TRX. Rats were intratracheally instilled with vehicle control, Dörentruper quartz (DQ12) or DQ12 coated with polyvinylpyridine N-oxide. At days 3, 7, 28, 90, 180 and 360 five animals per treatment group were sacrificed. Hallmarks of silicosis were assessed with Haematoxylin-eosin and Sirius Red stainings. Caspase-1 activity in the bronchoalveolar lavage and caspase-1 and IL-1ß localization in lung tissue were determined using Western blot and immunohistochemistry (IHC). RESULTS: Silica polymorphs triggered secretion of IL-1ß, bFGF and HMGB1 in a surface reactivity dependent manner. Inflammasome readouts linked with caspase-1 enzymatic activity were attenuated by TRX overexpression or treatment. At day 3 and 7 increased caspase-1 activity was detected in BALF of the DQ12 group and increased levels of caspase-1 and IL-1ß were observed with IHC in the DQ12 group compared to controls. DQ12 exposure revealed silicotic nodules at 180 and 360 days. Particle surface modification markedly attenuated the grade of inflammation and lymphocyte influx and attenuated the level of inflammasome activation, indicating that the development of silicosis and inflammasome activation is determined by crystalline silica surface reactivity. CONCLUSION: Our novel data indicate the pivotal role of surface reactivity of crystalline silica to activate the inflammasome in cultures of both epithelial cells and macrophages. Inhibitory capacity of the antioxidant TRX to inflammasome activation was evidenced. DQ12 quartz exposure induced acute and chronic functional activation of the inflammasome in the heterogeneous cell populations of the lung in associated with its crystalline surface reactivity.

Cigarette smoke extract induces a phenotypic shift in epithelial cells; involvement of HIF1α in mesenchymal transition.

In COPD, matrix remodeling contributes to airflow limitation. Recent evidence suggests that next to fibroblasts, the process of epithelial-mesenchymal transition can contribute to matrix remodeling. CSE has been shown to induce EMT in lung epithelial cells, but the signaling mechanisms involved are largely unknown and subject of this study. EMT was assessed in A549 and BEAS2B cells stimulated with CSE by qPCR, Western blotting and immunofluorescence for epithelial and mesenchymal markers, as were collagen production, cell adhesion and barrier integrity as functional endpoints. Involvement of TGF-ß and HIF1α signaling pathways were investigated. In addition, mouse models were used to examine the effects of CS on hypoxia signaling and of hypoxia per se on mesenchymal expression. CSE induced EMT characteristics in A549 and BEAS2B cells, evidenced by decreased expression of epithelial markers and a concomitant increase in mesenchymal marker expression after CSE exposure. Furthermore cells that underwent EMT showed increased production of collagen, decreased adhesion and disrupted barrier integrity. The induction of EMT was found to be independent of TGF-ß signaling. On the contrary, CS was able to induce hypoxic signaling in A549 and BEAS2B cells as well as in mice lung tissue. Importantly, HIF1α knock-down prevented induction of mesenchymal markers, increased collagen production and decreased adhesion after CSE exposure, data that are in line with the observed induction of mesenchymal marker expression by hypoxia in vitro and in vivo. Together these data provide evidence that both bronchial and alveolar epithelial cells undergo a functional phenotypic shift in response to CSE exposure which can contribute to increased collagen deposition in COPD lungs. Moreover, HIF1α signaling appears to play an important role in this process.

A comparative study of matrix remodeling in chronic models for COPD; mechanistic insights into the role of TNF-α.

Remodeling in chronic obstructive pulmonary disease (COPD) has at least two dimensions: small airway wall thickening and destruction of alveolar walls. Recently we showed comparable alterations of the extracellular matrix (ECM) compounds collagen, hyaluoran, and elastin in alveolar and small airway walls of COPD patients. The aim of this study was to characterize and assess similarities in alveolar and small airway wall matrix remodeling in chronic COPD models. From this comparative characterization of matrix remodeling we derived and elaborated underlying mechanisms to the matrix changes reported in COPD. Lung tissue sections of chronic models for COPD, either induced by exposure to cigarette smoke, chronic intratracheal lipopolysaccharide instillation, or local tumor necrosis factor (TNF) expression [surfactant protein C (SPC)-TNFα mice], were stained for elastin, collagen, and hyaluronan. Furthermore TNF-α matrix metalloproteinase (MMP)-2, -9, and -12 mRNA expression was analyzed using qPCR and localized using immunohistochemistry. Both collagen and hyaluronan were increased in alveolar and small airway walls of all three models. Interestingly, elastin contents were differentially affected, with a decrease in both alveolar and airway walls in SPC-TNFα mice. Furthermore TNF-α and MMP-2 and -9 mRNA and protein levels were found to be increased in alveolar walls and around airway walls only in SPC-TNFα mice. We show that only SPC-TNFα mice show changes in elastin remodeling that are comparable to what has been observed in COPD patients. This reveals that the SPC-TNFα model is a suitable model to study processes underlying matrix remodeling and in particular elastin breakdown as seen in COPD. Furthermore we indicate a possible role for MMP-2 and MMP-9 in the breakdown of elastin in airways and alveoli of SPC-TNFα mice.

Similar matrix alterations in alveolar and small airway walls of COPD patients.

BACKGROUND: Remodelling in COPD has at least two dimensions: small airway wall thickening and destruction of alveolar walls. Recent studies indicate that there is some similarity between alveolar and small airway wall matrix remodelling. The aim of this study was to characterise and assess similarities in alveolar and small airway wall matrix remodelling, and TGF-ß signalling in COPD patients of different GOLD stages. METHODS: Lung tissue sections of 14 smoking controls, 16 GOLD II and 19 GOLD IV patients were included and stained for elastin and collagens as well as hyaluronan, a glycosaminoglycan matrix component and pSMAD2. RESULTS: Elastin was significantly decreased in COPD patients not only in alveolar, but also in small airway walls. Interestingly, both collagen and hyaluronan were increased in alveolar as well as small airway walls. The matrix changes were highly comparable between GOLD stages, with collagen content in the alveolar wall increasing further in GOLD IV. A calculated remodelling index, defined as elastin divided over collagen and hyaluronan, was decreased significantly in GOLD II and further lowered in GOLD IV patients, suggesting that matrix component alterations are involved in progressive airflow limitation. Interestingly, there was a positive correlation present between the alveolar and small airway wall stainings of the matrix components, as well as for pSMAD2. No differences in pSMAD2 staining between controls and COPD patients were found. CONCLUSIONS: In conclusion, remodelling in the alveolar and small airway wall in COPD is markedly similar and already present in moderate COPD. Notably, alveolar collagen and a remodelling index relate to lung function.