The Leucocyte Telomere Length, GSTM1 and GSTT1 Null Genotypes and the Risk of Chronic Obstructive Pulmonary Disease

Chronic obstructive pulmonary disease (COPD) is characterized by chronic inflammation and oxidative stress both in the airways and blood and other organs. Elevated oxidative stress and inflammation have been reported to affect leucocyte telomere length (LTL). Glutathione S-transferase (GST) enzymes are a large family of xenobiotic-metabolizing enzymes that utilize different ROS products. We aimed to explore the link between GSTM1 and GSTT1 gene polymorphisms, LTL and COPD risk. For GSTM1, we genotyped 152 COPD patients and 131 non-affected controls; for GSTT1, we genotyped 149 COPD patients and 130 controls. We were able to assess TL for 91 patients and 88 controls. There was a significant difference in the GSTM1 null genotype frequency between the patients and controls (0.59 vs. 0.38, p ≤ 0.000), but such was not found for GSTT1 (p = 0.192). When combining both polymorphisms, we obtained a significantly greater presence of at least one null genotype among patients (0.12 vs. 0.05, p = 0.027). An association between GSTT1 and LTL was not found. COPD patients carrying the GSTM1 null genotype had shorter telomeres compared to those carrying the non-null genotype (15,720 bp vs. 22,442 bp, p = 0.008); as for the controls, it was the opposite (31,354 bp vs. 17,800 bp, p = 0.020). The significance in both groups remained when combining GSTM1 and GSTT1 (COPD (at least one null) 16,409 bp vs. COPD (non-null) 22,092 bp, p = 0.029; control (at least one null) 29,666 bp vs. control (non-null) 16,370 bp, p = 0.027). The total glutathione level in GSTM1 non-null controls was higher compared to the null genotype (15.39 ng/mL vs. 5.53 ng/mL, p = 0.002). In COPD patients, we found no association (p = 0.301). In conclusion, according to our results, GSTM1, but not GSTT1, null genotypes might play a role in leucocyte telomere shortening, and thus be involved in the pathogenesis of COPD

Advanced Respiratory Models for Hazard Assessment of Nanomaterials. Performance of Mono-, Co- and Tricultures

Advanced in vitro models are needed to support next-generation risk assessment (NGRA), moving from hazard assessment based mainly on animal studies to the application of new alternative methods (NAMs). Advanced models must be tested for hazard assessment of nanomaterials (NMs). The aim of this study was to perform an interlaboratory trial across two laboratories to test the robustness of and optimize a 3D lung model of human epithelial A549 cells cultivated at the air–liquid interface (ALI). Potential change in sensitivity in hazard identification when adding complexity, going from monocultures to co- and tricultures, was tested by including human endothelial cells EA.hy926 and differentiated monocytes dTHP-1. All models were exposed to NM-300K in an aerosol exposure system (VITROCELL® cloud-chamber). Cyto- and genotoxicity were measured by AlamarBlue and comet assay. Cellular uptake was investigated with transmission electron microscopy. The models were characterized by confocal microscopy and barrier function tested. We demonstrated that this advanced lung model is applicable for hazard assessment of NMs. The results point to a change in sensitivity of the model by adding complexity and to the importance of detailed protocols for robustness and reproducibility of advanced in vitro model

Advanced Respiratory Models for Hazard Assessment of Nanomaterials. Performance of Mono-, Co- and Tricultures

Advanced in vitro models are needed to support next-generation risk assessment (NGRA), moving from hazard assessment based mainly on animal studies to the application of new alternative methods (NAMs). Advanced models must be tested for hazard assessment of nanomaterials (NMs). The aim of this study was to perform an interlaboratory trial across two laboratories to test the robustness of and optimize a 3D lung model of human epithelial A549 cells cultivated at the air–liquid interface (ALI). Potential change in sensitivity in hazard identification when adding complexity, going from monocultures to co- and tricultures, was tested by including human endothelial cells EA.hy926 and differentiated monocytes dTHP-1. All models were exposed to NM-300K in an aerosol exposure system (VITROCELL® cloud-chamber). Cyto- and genotoxicity were measured by AlamarBlue and comet assay. Cellular uptake was investigated with transmission electron microscopy. The models were characterized by confocal microscopy and barrier function tested. We demonstrated that this advanced lung model is applicable for hazard assessment of NMs. The results point to a change in sensitivity of the model by adding complexity and to the importance of detailed protocols for robustness and reproducibility of advanced in vitro model

Advanced Respiratory Models for Hazard Assessment of Nanomaterials. Performance of Mono-, Co- and Tricultures

Advanced in vitro models are needed to support next-generation risk assessment (NGRA), moving from hazard assessment based mainly on animal studies to the application of new alternative methods (NAMs). Advanced models must be tested for hazard assessment of nanomaterials (NMs). The aim of this study was to perform an interlaboratory trial across two laboratories to test the robustness of and optimize a 3D lung model of human epithelial A549 cells cultivated at the air–liquid interface (ALI). Potential change in sensitivity in hazard identification when adding complexity, going from monocultures to co- and tricultures, was tested by including human endothelial cells EA.hy926 and differentiated monocytes dTHP-1. All models were exposed to NM-300K in an aerosol exposure system (VITROCELL® cloud-chamber). Cyto- and genotoxicity were measured by AlamarBlue and comet assay. Cellular uptake was investigated with transmission electron microscopy. The models were characterized by confocal microscopy and barrier function tested. We demonstrated that this advanced lung model is applicable for hazard assessment of NMs. The results point to a change in sensitivity of the model by adding complexity and to the importance of detailed protocols for robustness and reproducibility of advanced in vitro model

Advanced Respiratory Models for Hazard Assessment of Nanomaterials. Performance of Mono-, Co- and Tricultures

Advanced in vitro models are needed to support next-generation risk assessment (NGRA), moving from hazard assessment based mainly on animal studies to the application of new alternative methods (NAMs). Advanced models must be tested for hazard assessment of nanomaterials (NMs). The aim of this study was to perform an interlaboratory trial across two laboratories to test the robustness of and optimize a 3D lung model of human epithelial A549 cells cultivated at the air–liquid interface (ALI). Potential change in sensitivity in hazard identification when adding complexity, going from monocultures to co- and tricultures, was tested by including human endothelial cells EA.hy926 and differentiated monocytes dTHP-1. All models were exposed to NM-300K in an aerosol exposure system (VITROCELL® cloud-chamber). Cyto- and genotoxicity were measured by AlamarBlue and comet assay. Cellular uptake was investigated with transmission electron microscopy. The models were characterized by confocal microscopy and barrier function tested. We demonstrated that this advanced lung model is applicable for hazard assessment of NMs. The results point to a change in sensitivity of the model by adding complexity and to the importance of detailed protocols for robustness and reproducibility of advanced in vitro model

Multiwalled Carbon Nanotubes Induce Fibrosis and Telomere Length Alterations

Telomere shortening can result in cellular senescence and in increased level of genome instability, which are key events in numerous of cancer types. Despite this, few studies have focused on the effect of nanomaterial exposure on telomere length as a possible mechanism involved in nanomaterial-induced carcinogenesis. In this study, effects of exposure to multiwalled carbon nanotubes (MWCNT) on telomere length were investigated in mice exposed by intrapleural injection, as well as in human lung epithelial and mesothelial cell lines. In addition, cell cycle, apoptosis, and regulation of genes involved in DNA damage repair were assessed. Exposure to MWCNT led to severe fibrosis, infiltration of inflammatory cells in pleura, and mesothelial cell hyperplasia. These histological alterations were accompanied by deregulation of genes involved in fibrosis and immune cell recruitment, as well as a significant shortening of telomeres in the pleura and the lung. Assessment of key carcinogenic mechanisms in vitro confirmed that long-term exposure to the long MWCNT led to a prominent telomere shortening in epithelial cells, which coincided with G1-phase arrest and enhanced apoptosis. Altogether, our data show that telomere shortening resulting in cell cycle arrest and apoptosis may be an important mechanism in long MWCNT-induced inflammation and fibrosis

Transferability and reproducibility of exposed air-liquid interface co-culture lung models.

Overall, the results of the 7 participating laboratories are quite similar. After exposing Calu-3 alone and Calu-3 co-cultures with macrophages, no effects of lipopolysaccharide (LPS), quartz (DQ12) or titanium dioxide (TiO2) NM-105 particles on the cell viability and barrier integrity were detected. LPS exposure induced moderate cytokine release in the Calu-3 monoculture, albeit not statistically significant in most labs. In the co-culture models, most laboratories showed that LPS can significantly induce cytokine release (IL-6, IL-8 and TNF-α). The exposure to quartz and TiO2 particles did not induce a statistically significant increase in cytokine release in both cell models probably due to our relatively low deposited doses, which were inspired by in vivo dose levels. The intra- and inter-laboratory comparison study indicated acceptable interlaboratory variation for cell viability/toxicity (WST-1, LDH) and transepithelial electrical resistance, and relatively high inter-laboratory variation for cytokine production

Interlaboratory comparison of endotoxin contamination assessment of nanomaterials

Publisher Copyright: © 2024 The Royal Society of Chemistry.Endotoxin contamination is a significant hurdle to the translation of nanomaterials for biomedical applications. Multiple reports now describe that more than one-third of nanomaterials fail early pre-clinical assessment due to levels of endotoxin above regulatory requirements. Additionally, most immunological studies or in vivo studies testing nanomaterials in the literature lack inclusion of this assessment, which may lead to false-positive or false-negative results if high levels of the contaminant are present. The currently approved methods for endotoxin contamination assessment rely on enzymatic activity and wavelength absorbance as their endpoint, and many nanomaterials can interfere with such assays. For this reason, we devised an interlaboratory comparison of endotoxin contamination assessment for a range of nanomaterials to challenge the current international organization for standardization and pharmacopeia standards. Herein, we show that detected endotoxin levels could vary considerably between groups, and, in some instances, nanomaterials could both pass and fail regulatory endotoxin limits for medical devices depending on the group undertaking the assessment, all while passing all quality criteria standards. This work emphasises the requirement for multiple assays to fully assess the endotoxin levels in a nanomaterial and highlights the need for additional assays to be developed in this space.Peer reviewe

Transferability and reproducibility of exposed air-liquid interface co-culture lung models.

Overall, the results of the 7 participating laboratories are quite similar. After exposing Calu-3 alone and Calu-3 co-cultures with macrophages, no effects of lipopolysaccharide (LPS), quartz (DQ12) or titanium dioxide (TiO2) NM-105 particles on the cell viability and barrier integrity were detected. LPS exposure induced moderate cytokine release in the Calu-3 monoculture, albeit not statistically significant in most labs. In the co-culture models, most laboratories showed that LPS can significantly induce cytokine release (IL-6, IL-8 and TNF-α). The exposure to quartz and TiO2 particles did not induce a statistically significant increase in cytokine release in both cell models probably due to our relatively low deposited doses, which were inspired by in vivo dose levels. The intra- and inter-laboratory comparison study indicated acceptable interlaboratory variation for cell viability/toxicity (WST-1, LDH) and transepithelial electrical resistance, and relatively high inter-laboratory variation for cytokine production