Comparing the Toxicological Responses of Pulmonary Air-Liquid Interface Models upon Exposure to Differentially Treated Carbon Fibers.

In recent years, the use of carbon fibers (CFs) in various sectors of industry has been increasing. Despite the similarity of CF degradation products to other toxicologically relevant materials such as asbestos fibers and carbon nanotubes, a detailed toxicological evaluation of this class of material has yet to be performed. In this work, we exposed advanced air-liquid interface cell culture models of the human lung to CF. To simulate different stresses applied to CF throughout their life cycle, they were either mechanically (mCF) or thermo-mechanically pre-treated (tmCF). Different aspects of inhalation toxicity as well as their possible time-dependency were monitored. mCFs were found to induce a moderate inflammatory response, whereas tmCF elicited stronger inflammatory as well as apoptotic effects. Furthermore, thermal treatment changed the surface properties of the CF resulting in a presumed adhesion of the cells to the fiber fragments and subsequent cell loss. Triple-cultures encompassing epithelial, macrophage, and fibroblast cells stood out with an exceptionally high inflammatory response. Only a weak genotoxic effect was detected in the form of DNA strand breaks in mono- and co-cultures, with triple-cultures presenting a possible secondary genotoxicity. This work establishes CF fragments as a potentially harmful material and emphasizes the necessity of further toxicological assessment of existing and upcoming advanced CF-containing materials.

Gene Expression Profiling of Mono- and Co-Culture Models of the Respiratory Tract Exposed to Crystalline Quartz under Submerged and Air-Liquid Interface Conditions.

In vitro lung cell models like air-liquid interface (ALI) and 3D cell cultures have advanced greatly in recent years, being especially valuable for testing advanced materials (e.g., nanomaterials, fibrous substances) when considering inhalative exposure. Within this study, we established submerged and ALI cell culture models utilizing A549 cells as mono-cultures and co-cultures with differentiated THP-1 (dTHP-1), as well as mono-cultures of dTHP-1. After ALI and submerged exposures towards α-quartz particles (Min-U-Sil5), with depositions ranging from 15 to 60 µg/cm2, comparison was made with respect to their transcriptional cellular responses employing high-throughput RT-qPCR. A significant dose- and time-dependent induction of genes coding for inflammatory proteins, e.g., IL-1A, IL-1B, IL-6, IL-8, and CCL22, as well as genes associated with oxidative stress response such as SOD2, was observed, even more pronounced in co-cultures. Changes in the expression of similar genes were more pronounced under submerged conditions when compared to ALI exposure in the case of A549 mono-cultures. Hereby, the activation of the NF-κB signaling pathway and the NLRP3 inflammasome seem to play an important role. Regarding genotoxicity, neither DNA strand breaks in ALI cultivated cells nor a transcriptional response to DNA damage were observed. Altogether, the toxicological responses depended considerably on the cell culture model and exposure scenario, relevant to be considered to improve toxicological risk assessment.

Comparing α-Quartz-Induced Cytotoxicity and Interleukin-8 Release in Pulmonary Mono- and Co-Cultures Exposed under Submerged and Air-Liquid Interface Conditions.

The occupational exposure to particles such as crystalline quartz and its impact on the respiratory tract have been studied extensively in recent years. For hazard assessment, the development of physiologically more relevant in-vitro models, i.e., air-liquid interface (ALI) cell cultures, has greatly progressed. Within this study, pulmonary culture models employing A549 and differentiated THP-1 cells as mono-and co-cultures were investigated. The different cultures were exposed to α-quartz particles (Min-U-Sil5) with doses ranging from 15 to 66 µg/cm2 under submerged and ALI conditions and cytotoxicity as well as cytokine release were analyzed. No cytotoxicity was observed after ALI exposure. Contrarily, Min-U-Sil5 was cytotoxic at the highest dose in both submerged mono- and co-cultures. A concentration-dependent release of interleukin-8 was shown for both exposure types, which was overall stronger in co-cultures. Our findings showed considerable differences in the toxicological responses between ALI and submerged exposure and between mono- and co-cultures. A substantial influence of the presence or absence of serum in cell culture media was noted as well. Within this study, the submerged culture was revealed to be more sensitive. This shows the importance of considering different culture and exposure models and highlights the relevance of communication between different cell types for toxicological investigations.

Metabolic Activation of Benzo[a]pyrene by Human Tissue Organoid Cultures.

Organoids are 3D cultures that to some extent reproduce the structure, composition and function of the mammalian tissues from which they derive, thereby creating in vitro systems with more in vivo-like characteristics than 2D monocultures. Here, the ability of human organoids derived from normal gastric, pancreas, liver, colon and kidney tissues to metabolise the environmental carcinogen benzo[a]pyrene (BaP) was investigated. While organoids from the different tissues showed varied cytotoxic responses to BaP, with gastric and colon organoids being the most susceptible, the xenobiotic-metabolising enzyme (XME) genes, CYP1A1 and NQO1, were highly upregulated in all organoid types, with kidney organoids having the highest levels. Furthermore, the presence of two key metabolites, BaP-t-7,8-dihydrodiol and BaP-tetrol-l-1, was detected in all organoid types, confirming their ability to metabolise BaP. BaP bioactivation was confirmed both by the activation of the DNA damage response pathway (induction of p-p53, pCHK2, p21 and γ-H2AX) and by DNA adduct formation. Overall, pancreatic and undifferentiated liver organoids formed the highest levels of DNA adducts. Colon organoids had the lowest responses in DNA adduct and metabolite formation, as well as XME expression. Additionally, high-throughput RT-qPCR explored differences in gene expression between organoid types after BaP treatment. The results demonstrate the potential usefulness of organoids for studying environmental carcinogenesis and genetic toxicology.

Impact of Nanocomposite Combustion Aerosols on A549 Cells and a 3D Airway Model.

The use of nanomaterials incorporated into plastic products is increasing steadily. By using nano-scaled filling materials, thermoplastics, such as polyethylene (PE), take advantage of the unique properties of nanomaterials (NM). The life cycle of these so-called nanocomposites (NC) usually ends with energetic recovery. However, the toxicity of these aerosols, which may consist of released NM as well as combustion-generated volatile compounds, is not fully understood. Within this study, model nanocomposites consisting of a PE matrix and nano-scaled filling material (TiO2, CuO, carbon nano tubes (CNT)) were produced and subsequently incinerated using a lab-scale model burner. The combustion-generated aerosols were characterized with regard to particle release as well as compound composition. Subsequently, A549 cells and a reconstituted 3D lung cell culture model (MucilAir™, Epithelix) were exposed for 4 h to the respective aerosols. This approach enabled the parallel application of a complete aerosol, an aerosol under conditions of enhanced particle deposition using high voltage, and a filtered aerosol resulting in the sole gaseous phase. After 20 h post-incubation, cytotoxicity, inflammatory response (IL-8), transcriptional toxicity profiling, and genotoxicity were determined. Only the exposure toward combustion aerosols originated from PE-based materials induced cytotoxicity, genotoxicity, and transcriptional alterations in both cell models. In contrast, an inflammatory response in A549 cells was more evident after exposure toward aerosols of nano-scaled filler combustion, whereas the thermal decomposition of PE-based materials revealed an impaired IL-8 secretion. MucilAir™ tissue showed a pronounced inflammatory response after exposure to either combustion aerosols, except for nanocomposite combustion. In conclusion, this study supports the present knowledge on the release of nanomaterials after incineration of nano-enabled thermoplastics. Since in the case of PE-based combustion aerosols no major differences were evident between exposure to the complete aerosol and to the gaseous phase, adverse cellular effects could be deduced to the volatile organic compounds that are generated during incomplete combustion of NC.

Impact of Differentiated Macrophage-Like Cells on the Transcriptional Toxicity Profile of CuO Nanoparticles in Co-Cultured Lung Epithelial Cells.

To mimic more realistic lung tissue conditions, co-cultures of epithelial and immune cells are one comparatively easy-to-use option. To reveal the impact of immune cells on the mode of action (MoA) of CuO nanoparticles (NP) on epithelial cells, A549 cells as a model for epithelial cells have been cultured with or without differentiated THP-1 cells, as a model for macrophages. After 24 h of submerged incubation, cytotoxicity and transcriptional toxicity profiles were obtained and compared between the cell culture systems. Dose-dependent cytotoxicity was apparent starting from 8.0 µg/cm2 CuO NP. With regard to gene expression profiles, no differences between the cell models were observed concerning metal homeostasis, oxidative stress, and DNA damage, confirming the known MoA of CuO NP, i.e., endocytotic particle uptake, intracellular particle dissolution within lysosomes with subsequent metal ion deliberation, increased oxidative stress, and genotoxicity. However, applying a co-culture of epithelial and macrophage-like cells, CuO NP additionally provoked a pro-inflammatory response involving NLRP3 inflammasome and pro-inflammatory transcription factor activation. This study demonstrates that the application of this easy-to-use advanced in vitro model is able to extend the detection of cellular effects provoked by nanomaterials by an immunological response and emphasizes the use of such models to address a more comprehensive MoA.

Comparison of Metal-Based Nanoparticles and Nanowires: Solubility, Reactivity, Bioavailability and Cellular Toxicity.

While the toxicity of metal-based nanoparticles (NP) has been investigated in an increasing number of studies, little is known about metal-based fibrous materials, so-called nanowires (NWs). Within the present study, the physico-chemical properties of particulate and fibrous nanomaterials based on Cu, CuO, Ni, and Ag as well as TiO2 and CeO2 NP were characterized and compared with respect to abiotic metal ion release in different physiologically relevant media as well as acellular reactivity. While none of the materials was soluble at neutral pH in artificial alveolar fluid (AAF), Cu, CuO, and Ni-based materials displayed distinct dissolution under the acidic conditions found in artificial lysosomal fluids (ALF and PSF). Subsequently, four different cell lines were applied to compare cytotoxicity as well as intracellular metal ion release in the cytoplasm and nucleus. Both cytotoxicity and bioavailability reflected the acellular dissolution rates in physiological lysosomal media (pH 4.5); only Ag-based materials showed no or very low acellular solubility, but pronounced intracellular bioavailability and cytotoxicity, leading to particularly high concentrations in the nucleus. In conclusion, in spite of some quantitative differences, the intracellular bioavailability as well as toxicity is mostly driven by the respective metal and is less modulated by the shape of the respective NP or NW.

PARP1 catalytic variants reveal branching and chain length-specific functions of poly(ADP-ribose) in cellular physiology and stress response.

Poly(ADP-ribosyl)ation regulates numerous cellular processes like genome maintenance and cell death, thus providing protective functions but also contributing to several pathological conditions. Poly(ADP-ribose) (PAR) molecules exhibit a remarkable heterogeneity in chain lengths and branching frequencies, but the biological significance of this is basically unknown. To unravel structure-specific functions of PAR, we used PARP1 mutants producing PAR of different qualities, i.e. short and hypobranched (PARP1\G972R), short and moderately hyperbranched (PARP1\Y986S), or strongly hyperbranched PAR (PARP1\Y986H). By reconstituting HeLa PARP1 knockout cells, we demonstrate that PARP1\G972R negatively affects cellular endpoints, such as viability, cell cycle progression and genotoxic stress resistance. In contrast, PARP1\Y986S elicits only mild effects, suggesting that PAR branching compensates for short polymer length. Interestingly, PARP1\Y986H exhibits moderate beneficial effects on cell physiology. Furthermore, different PARP1 mutants have distinct effects on molecular processes, such as gene expression and protein localization dynamics of PARP1 itself, and of its downstream factor XRCC1. Finally, the biological relevance of PAR branching is emphasized by the fact that branching frequencies vary considerably during different phases of the DNA damage-induced PARylation reaction and between different mouse tissues. Taken together, this study reveals that PAR branching and chain length essentially affect cellular functions, which further supports the notion of a 'PAR code'.

Toxicity and Gene Expression Profiling of Copper- and Titanium-Based Nanoparticles Using Air-Liquid Interface Exposure.

To assess the toxicity of nanomaterials, most in vitro studies have been performed under submerged conditions, which do not reflect physiological conditions upon inhalation. An air-liquid interface (ALI) exposure may provide more reliable data on dosimetry and prevent interactions with cell culture media components. Therefore, an ALI exposure was combined with a high-throughput RT-qPCR approach to evaluate the toxicological potential of CuO and TiO2 nanoparticles (NP) in A549 cells. While TiO2 NP did not show any cytotoxicity or other effects compromising genomic stability up to 25.8 µg/cm2, CuO NP revealed a dose-dependent cytotoxicity, starting at 4.9 µg/cm2. Furthermore, CuO NP altered distinct gene expression patterns indicative for disturbed metal homeostasis, stress response, and DNA damage induction. Thus, induction of metal homeostasis associated genes (MT1X, MT2A) at 0.4 µg/cm2 and higher suggested uptake and intracellular dissolution of CuO NP, which was verified by a dose-dependent increase in intracellular copper concentration. Starting at 4.9 µg/cm2, oxidative stress markers (HMOX1, HSPA1A) were induced dose-dependently, supported by elevated ROS levels. Furthermore, a dose-dependent induction of genes associated with DNA damage response (DDIT3, GADD45A) was observed, in concordance with an increase in DNA strand breaks. Finally, transcriptional data suggested the induction of apoptosis at high doses, while flow cytometric analysis revealed increased numbers of either late apoptotic or necrotic cells and clearly necrotic cells at the highest concentrations. Thus, an ALI cell culture system was successfully combined with a comprehensive high-throughput RT-qPCR system, allowing the quantification of NP deposition and their impact on genomic stability. For CuO NP, in principle the data confirm observations made under submerged conditions with respect to intracellular copper ion release, as well as oxidative and genotoxic stress response. However, the results derived from ALI exposure allow the assessment of dose-response-relationships as well as the comparison of relative toxic potencies of different NP.

The C-terminal domain of p53 orchestrates the interplay between non-covalent and covalent poly(ADP-ribosyl)ation of p53 by PARP1.

The post-translational modification poly(ADP-ribosyl)ation (PARylation) plays key roles in genome maintenance and transcription. Both non-covalent poly(ADP-ribose) binding and covalent PARylation control protein functions, however, it is unknown how the two modes of modification crosstalk mechanistically. Employing the tumor suppressor p53 as a model substrate, this study provides detailed insights into the interplay between non-covalent and covalent PARylation and unravels its functional significance in the regulation of p53. We reveal that the multifunctional C-terminal domain (CTD) of p53 acts as the central hub in the PARylation-dependent regulation of p53. Specifically, p53 bound to auto-PARylated PARP1 via highly specific non-covalent PAR-CTD interaction, which conveyed target specificity for its covalent PARylation by PARP1. Strikingly, fusing the p53-CTD to a protein that is normally not PARylated, renders this a target for covalent PARylation as well. Functional studies revealed that the p53-PAR interaction had substantial implications on molecular and cellular levels. Thus, PAR significantly influenced the complex p53-DNA binding properties and controlled p53 functions, with major implications on the p53-dependent interactome, transcription, and replication-associated recombination. Remarkably, this mechanism potentially also applies to other PARylation targets, since a bioinformatics analysis revealed that CTD-like regions are highly enriched in the PARylated proteome.