Characterization of the internal working-life exposome using minimally and non-invasive sampling methods - a narrative review.

During recent years, we are moving away from the 'one exposure, one disease'-approach in occupational settings and towards a more comprehensive approach, taking into account the totality of exposures during a life course by using an exposome approach. Taking an exposome approach however is accompanied by many challenges, one of which, for example, relates to the collection of biological samples. Methods used for sample collection in occupational exposome studies should ideally be minimally invasive, while at the same time sensitive, and enable meaningful repeated sampling in a large population and over a longer time period. This might be hampered in specific situations e.g., people working in remote areas, during pandemics or with flexible work hours. In these situations, using self-sampling techniques might offer a solution. Therefore, our aim was to identify existing self-sampling techniques and to evaluate the applicability of these techniques in an occupational exposome context by conducting a literature review. We here present an overview of current self-sampling methodologies used to characterize the internal exposome. In addition, the use of different biological matrices was evaluated and subdivided based on their level of invasiveness and applicability in an occupational exposome context. In conclusion, this review and the overview of self-sampling techniques presented herein can serve as a guide in the design of future (occupational) exposome studies while circumventing sample collection challenges associated with exposome studies.

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

BACKGROUND: The establishment of reliable and robust in vitro models for hazard assessment, a prerequisite for moving away from animal testing, requires the evaluation of model transferability and reproducibility. Lung models that can be exposed via the air, by means of an air-liquid interface (ALI) are promising in vitro models for evaluating the safety of nanomaterials (NMs) after inhalation exposure. We performed an inter-laboratory comparison study to evaluate the transferability and reproducibility of a lung model consisting of the human bronchial cell line Calu-3 as a monoculture and, to increase the physiologic relevance of the model, also as a co-culture with macrophages (either derived from the THP-1 monocyte cell line or from human blood monocytes). The lung model was exposed to NMs using the VITROCELL® Cloud12 system at physiologically relevant dose levels. RESULTS: 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. CONCLUSION: The transferability and reproducibility of a lung co-culture model and its exposure to aerosolized particles at the ALI were evaluated and recommendations were provided for performing inter-laboratory comparison studies. Although the results are promising, optimizations of the lung model (including more sensitive read-outs) and/or selection of higher deposited doses are needed to enhance its predictive value before it may be taken further towards a possible OECD guideline.

High aspect ratio nanomaterial-induced macrophage polarization is mediated by changes in miRNA levels.

Introduction: Inhalation of nanomaterials may induce inflammation in the lung which if left unresolved can manifest in pulmonary fibrosis. In these processes, alveolar macrophages have an essential role and timely modulation of the macrophage phenotype is imperative in the onset and resolution of inflammatory responses. This study aimed to investigate, the immunomodulating properties of two industrially relevant high aspect ratio nanomaterials, namely nanocellulose and multiwalled carbon nanotubes (MWCNT), in an alveolar macrophage model. Methods: MH-S alveolar macrophages were exposed at air-liquid interface to cellulose nanocrystals (CNC), cellulose nanofibers (CNF) and two MWCNT (NM-400 and NM-401). Following exposure, changes in macrophage polarization markers and secretion of inflammatory cytokines were analyzed. Furthermore, the potential contribution of epigenetic regulation in nanomaterial-induced macrophage polarization was investigated by assessing changes in epigenetic regulatory enzymes, miRNAs, and rRNA modifications. Results: Our data illustrate that the investigated nanomaterials trigger phenotypic changes in alveolar macrophages, where CNF exposure leads to enhanced M1 phenotype and MWCNT promotes M2 phenotype. Furthermore, MWCNT exposure induced more prominent epigenetic regulatory events with changes in the expression of histone modification and DNA methylation enzymes as well as in miRNA transcript levels. MWCNT-enhanced changes in the macrophage phenotype were correlated with prominent downregulation of the histone methyltransferases Kmt2a and Smyd5 and histone deacetylases Hdac4, Hdac9 and Sirt1 indicating that both histone methylation and acetylation events may be critical in the Th2 responses to MWCNT. Furthermore, MWCNT as well as CNF exposure led to altered miRNA levels, where miR-155-5p, miR-16-1-3p, miR-25-3p, and miR-27a-5p were significantly regulated by both materials. PANTHER pathway analysis of the identified miRNA targets showed that both materials affected growth factor (PDGF, EGF and FGF), Ras/MAPKs, CCKR, GnRH-R, integrin, and endothelin signaling pathways. These pathways are important in inflammation or in the activation, polarization, migration, and regulation of phagocytic capacity of macrophages. In addition, pathways involved in interleukin, WNT and TGFB signaling were highly enriched following MWCNT exposure. Conclusion: Together, these data support the importance of macrophage phenotypic changes in the onset and resolution of inflammation and identify epigenetic patterns in macrophages which may be critical in nanomaterial-induced inflammation and fibrosis.

An inter-laboratory effort to harmonize the cell-delivered in vitro dose of aerosolized materials.

Air-liquid interface (ALI) lung cell models cultured on permeable transwell inserts are increasingly used for respiratory hazard assessment requiring controlled aerosolization and deposition of any material on ALI cells. The approach presented herein aimed to assess the transwell insert-delivered dose of aerosolized materials using the VITROCELL® Cloud12 system, a commercially available aerosol-cell exposure system. An inter-laboratory comparison study was conducted with seven European partners having different levels of experience with the VITROCELL® Cloud12. A standard operating procedure (SOP) was developed and applied by all partners for aerosolized delivery of materials, i.e., a water-soluble molecular substance (fluorescence-spiked salt) and two poorly soluble particles, crystalline silica quartz (DQ12) and titanium dioxide nanoparticles (TiO2 NM-105). The material dose delivered to transwell inserts was quantified with spectrofluorometry (fluorescein) and with the quartz crystal microbalance (QCM) integrated in the VITROCELL® Cloud12 system. The shape and agglomeration state of the deposited particles were confirmed with transmission electron microscopy (TEM). Inter-laboratory comparison of the device-specific performance was conducted in two steps, first for molecular substances (fluorescein-spiked salt), and then for particles. Device- and/or handling-specific differences in aerosol deposition of VITROCELL® Cloud12 systems were characterized in terms of the so-called deposition factor (DF), which allows for prediction of the transwell insert-deposited particle dose from the particle concentration in the aerosolized suspension. Albeit DF varied between the different labs from 0.39 to 0.87 (mean (coefficient of variation (CV)): 0.64 (28%)), the QCM of each VITROCELL® Cloud 12 system accurately measured the respective transwell insert-deposited dose. Aerosolized delivery of DQ12 and TiO2 NM-105 particles showed good linearity (R2 > 0.95) between particle concentration of the aerosolized suspension and QCM-determined insert-delivered particle dose. The VITROCELL® Cloud 12 performance for DQ12 particles was identical to that for fluorescein-spiked salt, i.e., the ratio of measured and salt-predicted dose was 1.0 (29%). On the other hand, a ca. 2-fold reduced dose was observed for TiO2 NM-105 (0.54 (41%)), which was likely due to partial retention of TiO2 NM-105 agglomerates in the vibrating mesh nebulizer of the VITROCELL® Cloud12. This inter-laboratory comparison demonstrates that the QCM integrated in the VITROCELL® Cloud 12 is a reliable tool for dosimetry, which accounts for potential variations of the transwell insert-delivered dose due to device-, handling- and/or material-specific effects. With the detailed protocol presented herein, all seven partner laboratories were able to demonstrate dose-controlled aerosolization of material suspensions using the VITROCELL® Cloud12 exposure system at dose levels relevant for observing in vitro hazard responses. This is an important step towards regulatory approved implementation of ALI lung cell cultures for in vitro hazard assessment of aerosolized materials.

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.

In vitro-in vivo correlations of pulmonary inflammogenicity and genotoxicity of MWCNT.

BACKGROUND: Multi-walled carbon nanotubes (MWCNT) have received attention due to extraordinary properties, resulting in concerns for occupational health and safety. Costs and ethical concerns of animal testing drive a need for in vitro models with predictive power in respiratory toxicity. The aim of this study was to assess pro-inflammatory response (Interleukin-8 expression, IL-8) and genotoxicity (DNA strand breaks) caused by MWCNT with different physicochemical properties in different pulmonary cell models and correlate these to previously published in vivo data. Seven MWCNT were selected; two long/thick (NRCWE-006/Mitsui-7 and NM-401), two short/thin (NM-400 and NM-403), a pristine (NRCWE-040) and two surface modified; hydroxylated (NRCWE-041) and carboxylated (NRCWE-042). Carbon black Printex90 (CB) was included as benchmark material. Human alveolar epithelial cells (A549) and monocyte-derived macrophages (THP-1a) were exposed to nanomaterials (NM) in submerged conditions, and two materials (NM-400 and NM-401) in co-cultures of A549/THP-1a and lung fibroblasts (WI-38) in an air-liquid interface (ALI) system. Effective doses were quantified by thermo-gravimetric-mass spectrometry analysis (TGA-MS). To compare genotoxicity in vitro and in vivo, we developed a scoring system based on a categorization of effects into standard deviation (SD) units (< 1, 1, 2, 3 or 4 standard deviation increases) for the increasing genotoxicity. RESULTS: Effective doses were shown to be 25 to 53%, and 21 to 57% of the doses administered to A549 and THP-1a, respectively. In submerged conditions (A549 and THP-1a cells), all NM induced dose-dependent IL-8 expression. NM-401 and NRCWE-006 caused the strongest pro-inflammatory response. In the ALI-exposed co-culture, only NM-401 caused increased IL-8 expression, and no DNA strand breaks were observed. Strong correlations were found between in vitro and in vivo inflammation when doses were normalized by surface area (also proxy for diameter and length). Significantly increased DNA damage was found for all MWCNT in THP-1a cells, and for short MWCNT in A549 cells. A concordance in genotoxicity of 83% was obtained between THP-1a cells and broncho-alveolar lavaged (BAL) cells. CONCLUSION: This study shows correlations of pro-inflammatory potential in A549 and THP-1a cells with neutrophil influx in mice, and concordance in genotoxic response between THP-1a cells and BAL cells, for seven MWCNT.

Long-Term Exposure to Nanosized TiO2 Triggers Stress Responses and Cell Death Pathways in Pulmonary Epithelial Cells.

There is little in vitro data available on long-term effects of TiO2 exposure. Such data are important for improving the understanding of underlying mechanisms of adverse health effects of TiO2. Here, we exposed pulmonary epithelial cells to two doses (0.96 and 1.92 µg/cm2) of TiO2 for 13 weeks and effects on cell cycle and cell death mechanisms, i.e., apoptosis and autophagy were determined after 4, 8 and 13 weeks of exposure. Changes in telomere length, cellular protein levels and lipid classes were also analyzed at 13 weeks of exposure. We observed that the TiO2 exposure increased the fraction of cells in G1-phase and reduced the fraction of cells in G2-phase, which was accompanied by an increase in the fraction of late apoptotic/necrotic cells. This corresponded with an induced expression of key apoptotic proteins i.e., BAD and BAX, and an accumulation of several lipid classes involved in cellular stress and apoptosis. These findings were further supported by quantitative proteome profiling data showing an increase in proteins involved in cell stress and genomic maintenance pathways following TiO2 exposure. Altogether, we suggest that cell stress response and cell death pathways may be important molecular events in long-term health effects of TiO2.

The matri-cellular proteins 'cysteine-rich, angiogenic-inducer, 61' and 'connective tissue growth factor' are regulated in experimentally-induced sepsis with multiple organ dysfunction.

Organ failure is a severe complication in sepsis for which the pathophysiology remains incompletely understood. Recently, the matri-cellular cysteine-rich, angiogenic induced, 61 (Cyr61/CCN1); connective tissue growth factor (Ctgf/CCN2); and nephroblastoma overexpressed gene (Nov/CCN3) (CCN)-protein family have been attributed organ-protective properties. Their expression is sensitive to mediators of sepsis pathophysiology but a potential role in sepsis remains elusive. To provide an initial assessment, 50 rats were subjected to 18 h of cecal-ligation and puncture or sham operation. Hepatic and pulmonary CCN1 mRNA displayed an average 7.4- and 3.3-fold induction, while its cardiac expression was unchanged. The changes coincided with excessive hepatic and pulmonary inflammatory gene activation and a restricted cardiac inflammation. Furthermore, hepatocytes displayed a dosage-dependent CCN1 mRNA response in vitro, supporting a cytokine-mediated CCN1 regulation in sepsis. CCN2 mRNA was 2.2-fold induced in the liver, while 2.0-fold and 1.4-fold repressed in the heart and lung. Meanwhile, it did not respond to TNF-α exposure in vitro, which indicates different means of regulation than for CCN1. Taken together, this study provides the first evidence for multi-organ regulation of CCN1 and CCN2 in early stages of sepsis, and implies the eruption of inflammatory mediators as a potential mechanism behind the observed CCN1 regulation.