A novel human 3D lung microtissue model for nanoparticle-induced cell-matrix alterations.

BACKGROUND: Multi-walled carbon nanotubes (MWCNT) have been shown to elicit the release of inflammatory and pro-fibrotic mediators, as well as histopathological changes in lungs of exposed animals. Current standards for testing MWCNTs and other nanoparticles (NPs) rely on low-throughput in vivo studies to assess acute and chronic toxicity and potential hazard to humans. Several alternative testing approaches utilizing two-dimensional (2D) in vitro assays to screen engineered NPs have reported conflicting results between in vitro and in vivo assays. Compared to conventional 2D in vitro or in vivo animal model systems, three-dimensional (3D) in vitro platforms have been shown to more closely recapitulate human physiology, providing a relevant, more efficient strategy for evaluating acute toxicity and chronic outcomes in a tiered nanomaterial toxicity testing paradigm. RESULTS: As inhalation is an important route of nanomaterial exposure, human lung fibroblasts and epithelial cells were co-cultured with macrophages to form scaffold-free 3D lung microtissues. Microtissues were exposed to multi-walled carbon nanotubes, M120 carbon black nanoparticles or crocidolite asbestos fibers for 4 or 7 days, then collected for characterization of microtissue viability, tissue morphology, and expression of genes and selected proteins associated with inflammation and extracellular matrix remodeling. Our data demonstrate the utility of 3D microtissues in predicting chronic pulmonary endpoints following exposure to MWCNTs or asbestos fibers. These test nanomaterials were incorporated into 3D human lung microtissues as visualized using light microscopy. Differential expression of genes involved in acute inflammation and extracellular matrix remodeling was detected using PCR arrays and confirmed using qRT-PCR analysis and Luminex assays of selected genes and proteins. CONCLUSION: 3D lung microtissues provide an alternative testing platform for assessing nanomaterial-induced cell-matrix alterations and delineation of toxicity pathways, moving towards a more predictive and physiologically relevant approach for in vitro NP toxicity testing.

A 3D fish liver model for aquatic toxicology: Morphological changes and Cyp1a induction in PLHC-1 microtissues after repeated benzo(a)pyrene exposures.

To identify the potential environmental impacts of aquatic pollutants, rapid and sensitive screening tools are needed to assess adaptive and toxic responses. This study characterizes a novel fish liver microtissue model, produced with the cell line PLHC-1, as an in vitro aquatic toxicity testing platform. These 3D microtissues remain viable and stable throughout the 8-day testing period and relative to 2D monolayers, show increased basal expression of the xenobiotic metabolizing enzyme cytochrome P450 1A (Cyp1a). To evaluate pulsed, low-dose exposures at environmentally relevant concentrations, microtissue responsiveness to the model toxicant benzo(a)pyrene was assessed after single and repeated exposures for determination of both immediate and persistent effects. Significant induction of Cyp1a gene and protein expression was detected after a single 24h exposure to as little as 1nM benzo(a)pyrene, and after a 24h recovery period, Cyp1a expression declined in a dose-dependent manner. However, cell death continued to increase during the recovery period and alterations in microtissue architecture occurred at higher concentrations. To evaluate a pulsed or repeated exposure scenario, microtissues were exposed to benzo(a)pyrene, allowed to recover, then exposed a second time for 24h. Following pre-exposure to benzo(a)pyrene, cyp1a expression remained equally inducible and the pattern and level of Cyp1a protein response to a second exposure were comparable. However, pre-exposure to 1µM or 5µM of benzo(a)pyrene resulted in increased cell death, greater disruption of microtissue architecture, and alterations in cell morphology. Together, this study demonstrates the capabilities of this PLHC-1 microtissue model for sensitive assessment of liver toxicants over time and following single and repeated exposures.

Hypothalamic Sirt1 regulates food intake in a rodent model system.

Sirt1 is an evolutionarily conserved NAD(+) dependent deacetylase involved in a wide range of processes including cellular differentiation, apoptosis, as well as metabolism, and aging. In this study, we investigated the role of hypothalamic Sirt1 in energy balance. Pharmacological inhibition or siRNA mediated knock down of hypothalamic Sirt1 showed to decrease food intake and body weight gain. Central administration of a specific melanocortin antagonist, SHU9119, reversed the anorectic effect of hypothalamic Sirt1 inhibition, suggesting that Sirt1 regulates food intake through the central melanocortin signaling. We also showed that fasting increases hypothalamic Sirt1 expression and decreases FoxO1 (Forkhead transcription factor) acetylation suggesting that Sirt1 regulates the central melanocortin system in a FoxO1 dependent manner. In addition, hypothalamic Sirt1 showed to regulate S6K signaling such that inhibition of the fasting induced Sirt1 activity results in up-regulation of the S6K pathway. Thus, this is the first study providing a novel role for the hypothalamic Sirt1 in the regulation of food intake and body weight. Given the role of Sirt1 in several peripheral tissues and hypothalamus, potential therapies centered on Sirt1 regulation might provide promising therapies in the treatment of metabolic diseases including obesity.

Accelerated progression of asbestos-induced mesotheliomas in heterozygous p53+/- mice.

Asbestos fibers produce diffuse malignant mesotheliomas in chronic rodent inhalation assays or after direct intrapleural or intraperitoneal injection. In vitro models have provided evidence that asbestos fibers are genotoxic carcinogens that can directly or indirectly generate reactive oxygen- and nitrogen-derived species that cause DNA damage. Heterozygous p53+/- mice show an increased incidence and reduced latency of malignant mesotheliomas induced by weekly intraperitoneal injections of crocidolite asbestos fibers. In this study, we investigated whether loss of heterozygosity (LOH) at the p53 tumor-suppressor gene locus contributes to accelerated tumor progression. LOH was found in 50% of the tumors produced in heterozygous p53+/- mice. In contrast to tumors that arise in p53+/+ mice or those that retained one p53 allele, LOH was associated with large tumor masses with central areas of necrosis, local invasion, and penetration of lymphatics. Increased tumor size was not associated with increased levels of cell proliferation as determined by BrdU incorporation, but it was correlated with a reduction in apoptosis as determined morphologically and by the TUNEL assay. Wild-type p53 protein is essential for cell cycle arrest in response to DNA damage and in maintenance of genomic stability. Cell lines established from tumors that showed LOH at the p53 tumor-suppressor gene locus were nearly tetraploid. These results suggest that p53 haplo-insufficiency sensitizes mice to the clastogenic or aneuploidogenic effects of crocidolite asbestos fibers, resulting in a shorter latent period. As solid tumors develop, spontaneous loss of the wild-type allele accompanied by decreased apoptosis and genetic instability is associated with accelerated tumor growth, invasion, and lymphatic dissemination.