Distributions for time, interspecies and intraspecies extrapolation for deriving occupational exposure limits.

This work aimed at improving the empirical database of time (i.e., exposure duration), interspecies and intraspecies extrapolation when deriving occupational exposure limits (OELs). For each extrapolation step, a distribution was derived, which can be used to model the associated uncertainties. For time and interspecies extrapolation, distributions of ratios of dose descriptors were derived from studies of different length or species. National Toxicology Program (NTP) study data were manually assessed, and data from REACH (Registration, Evaluation and Authorisation of Chemicals) registration dossiers were evaluated semi-automatically. Intraspecies extrapolation was investigated by compiling published studies on human toxicokinetic and toxicodynamic variability. A new database was established for toxicokinetic differences in interindividual susceptibility, including many inhalation studies. Using NTP data produced more reliable results than using REACH data. The geometric mean (GM) for time extrapolation subacute/chronic agreed with previous evaluations (GM = 4.11), whereas the GM for subchronic/chronic extrapolation was slightly higher (GM = 2.93) than the GMs found by others. No significant differences were observed between systemically and locally acting substances. Observed interspecies differences confirmed the suitability of allometric scaling, with the derived distribution describing remaining uncertainty. Distributions of intraspecies variability at the 1% and 5% incidence level had medians of 7.25 and 3.56, respectively. When compared with assessment factors (AFs) currently used in the EU, probabilities that these AFs are protective enough span a wide range from 10% to 95%, depending on the extrapolation step. These results help to select AFs in a transparent and informed way and, by allowing to compare protection levels achieved, to harmonise methods for deriving OELs.

Derivation of occupational exposure limits: Differences in methods and protection levels.

Frameworks for deriving occupational exposure limits (OELs) and OEL-analogue values (such as derived-no-effect levels [DNELs]) in various regulatory areas in the EU and at national level in Germany were analysed. Reasons for differences between frameworks and possible means of improving transparency and harmonisation were identified. Differences between assessment factors used for deriving exposure limits proved to be one important reason for diverging numerical values. Distributions for exposure time, interspecies and intraspecies extrapolation were combined by probabilistic methods and compared with default values of assessment factors used in the various OEL frameworks in order to investigate protection levels. In a subchronic inhalation study showing local effects in the respiratory tract, the probability that assessment factors were sufficiently high to protect 99% and 95% of the target population (workers) from adverse effects varied considerably from 9% to 71% and 17% to 87%, respectively, between the frameworks. All steps of the derivation process, including the uncertainty associated with the point of departure (POD), were further analysed with two examples of full probabilistic assessments. It is proposed that benchmark modelling should be the method of choice for deriving PODs and that all OEL frameworks should provide detailed guidance documents and clearly define their protection goals by stating the proportion of the exposed population the OEL aims to cover and the probability with which they intend to provide protection from adverse effects. Harmonisation can be achieved by agreeing on the way to perform the methodological steps for deriving OELs and on common protection goals.

Influence of wood species on toxicity of log-wood stove combustion aerosols: a parallel animal and air-liquid interface cell exposure study on spruce and pine smoke.

BACKGROUND: Wood combustion emissions have been studied previously either by in vitro or in vivo models using collected particles, yet most studies have neglected gaseous compounds. Furthermore, a more accurate and holistic view of the toxicity of aerosols can be gained with parallel in vitro and in vivo studies using direct exposure methods. Moreover, modern exposure techniques such as air-liquid interface (ALI) exposures enable better assessment of the toxicity of the applied aerosols than, for example, the previous state-of-the-art submerged cell exposure techniques. METHODS: We used three different ALI exposure systems in parallel to study the toxicological effects of spruce and pine combustion emissions in human alveolar epithelial (A549) and murine macrophage (RAW264.7) cell lines. A whole-body mouse inhalation system was also used to expose C57BL/6 J mice to aerosol emissions. Moreover, gaseous and particulate fractions were studied separately in one of the cell exposure systems. After exposure, the cells and animals were measured for various parameters of cytotoxicity, inflammation, genotoxicity, transcriptome and proteome. RESULTS: We found that diluted (1:15) exposure pine combustion emissions (PM1 mass 7.7 ± 6.5 mg m- 3, 41 mg MJ- 1) contained, on average, more PM and polycyclic aromatic hydrocarbons (PAHs) than spruce (PM1 mass 4.3 ± 5.1 mg m- 3, 26 mg MJ- 1) emissions, which instead showed a higher concentration of inorganic metals in the emission aerosol. Both A549 cells and mice exposed to these emissions showed low levels of inflammation but significantly increased genotoxicity. Gaseous emission compounds produced similar genotoxicity and a higher inflammatory response than the corresponding complete combustion emission in A549 cells. Systems biology approaches supported the findings, but we detected differing responses between in vivo and in vitro experiments. CONCLUSIONS: Comprehensive in vitro and in vivo exposure studies with emission characterization and systems biology approaches revealed further information on the effects of combustion aerosol toxicity than could be achieved with either method alone. Interestingly, in vitro and in vivo exposures showed the opposite order of the highest DNA damage. In vitro measurements also indicated that the gaseous fraction of emission aerosols may be more important in causing adverse toxicological effects. Combustion aerosols of different wood species result in mild but aerosol specific in vitro and in vivo effects.

Ultra-Fast Synthesis of Multivalent Radical Nanoparticles by Ring-Opening Metathesis Polymerization-Induced Self-Assembly.

We report the straightforward, time-efficient synthesis of radical core-shell nanoparticles (NPs) by polymerization-induced self-assembly. A nitroxide-containing hydrophilic macromolecular precursor was prepared by ring-opening metathesis copolymerization of norbornenyl derivatives of TEMPO and oligoethylene glycol and was chain-extended in situ with norbornene in ethanolic solution, leading to simultaneous amphiphilic block copolymer formation and self-assembly. Without any intermediate purification from the monomers to the block copolymers, radical NPs with tunable diameters ranging from 10 to 110 nm are obtained within minutes at room temperature. The high activity of the radical NPs as chemoselective and homogeneous, yet readily recyclable catalysts is demonstrated through oxidation of a variety of alcohols and recovery by simple centrifugation. Furthermore, the NPs show biocompatibility and antioxidant activity in vitro.

Toxicity of wood smoke particles in human A549 lung epithelial cells: the role of PAHs, soot and zinc.

Indoor air pollution is associated with increased morbidity and mortality. Specifically, the health impact of emissions from domestic burning of biomass and coal is most relevant and is estimated to contribute to over 4 million premature deaths per year worldwide. Wood is the main fuel source for biomass combustion and the shift towards renewable energy sources will further increase emissions from wood combustion even in developed countries. However, little is known about the constituents of wood smoke and biological mechanisms that are responsible for adverse health effects. We exposed A549 lung epithelial cells to collected wood smoke particles and found an increase in cellular reactive oxygen species as well as a response to bioavailable polycyclic aromatic hydrocarbons. In contrast, cell vitality and regulation of the pro-inflammatory cytokine interleukin-8 were not affected. Using a candidate approach, we could recapitulate WSP toxicity by the combined actions of its constituents soot, metals and PAHs. The soot fraction and metals were found to be the most important factors for ROS formation, whereas the PAH response can be mimicked by the model PAH benzo[a]pyrene. Strikingly, PAHs adsorbed to WSPs were even more potent in activating target gene expression than B[a]P individually applied in suspension. As PAHs initiate multiple adverse outcome pathways and are prominent carcinogens, their role as key pollutants in wood smoke and its health effects warrants further investigation. The presented results suggest that each of the investigated constituents soot, metals and PAHs are major contributors to WSP toxicity. Mitigation strategies to prevent adverse health effects of wood combustion should therefore not only aim at reducing the emitted soot and PAHs but also the metal content, through the use of more efficient combustion appliances, and particle precipitation techniques, respectively.

Systems toxicology of complex wood combustion aerosol reveals gaseous carbonyl compounds as critical constituents.

Epidemiological studies identified air pollution as one of the prime causes for human morbidity and mortality, due to harmful effects mainly on the cardiovascular and respiratory systems. Damage to the lung leads to several severe diseases such as fibrosis, chronic obstructive pulmonary disease and cancer. Noxious environmental aerosols are comprised of a gas and particulate phase representing highly complex chemical mixtures composed of myriads of compounds. Although some critical pollutants, foremost particulate matter (PM), could be linked to adverse health effects, a comprehensive understanding of relevant biological mechanisms and detrimental aerosol constituents is still lacking. Here, we employed a systems toxicology approach focusing on wood combustion, an important source for air pollution, and demonstrate a key role of the gas phase, specifically carbonyls, in driving adverse effects. Transcriptional profiling and biochemical analysis of human lung cells exposed at the air-liquid-interface determined DNA damage and stress response, as well as perturbation of cellular metabolism, as major key events. Connectivity mapping revealed a high similarity of gene expression signatures induced by wood smoke and agents prompting DNA-protein crosslinks (DPCs). Indeed, various gaseous aldehydes were detected in wood smoke, which promote DPCs, initiate similar genomic responses and are responsible for DNA damage provoked by wood smoke. Hence, systems toxicology enables the discovery of critical constituents of complex mixtures i.e. aerosols and highlights the role of carbonyls on top of particulate matter as an important health hazard.

A chemical probe for BAG1 targets androgen receptor-positive prostate cancer through oxidative stress signaling pathway.

BAG1 is a family of polypeptides with a conserved C-terminal BAG domain that functions as a nucleotide exchange factor for the molecular chaperone HSP70. BAG1 proteins also control several signaling processes including proteostasis, apoptosis, and transcription. The largest isoform, BAG1L, controls the activity of the androgen receptor (AR) and is upregulated in prostate cancer. Here, we show that BAG1L regulates AR dynamics in the nucleus and its ablation attenuates AR target gene expression especially those involved in oxidative stress and metabolism. We show that a small molecule, A4B17, that targets the BAG domain downregulates AR target genes similar to a complete BAG1L knockout and upregulates the expression of oxidative stress-induced genes involved in cell death. Furthermore, A4B17 outperformed the clinically approved antagonist enzalutamide in inhibiting cell proliferation and prostate tumor development in a mouse xenograft model. BAG1 inhibitors therefore offer unique opportunities for antagonizing AR action and prostate cancer growth.

ELIXIR and Toxicology: a community in development.

Toxicology has been an active research field for many decades, with academic, industrial and government involvement. Modern omics and computational approaches are changing the field, from merely disease-specific observational models into target-specific predictive models. Traditionally, toxicology has strong links with other fields such as biology, chemistry, pharmacology and medicine. With the rise of synthetic and new engineered materials, alongside ongoing prioritisation needs in chemical risk assessment for existing chemicals, early predictive evaluations are becoming of utmost importance to both scientific and regulatory purposes. ELIXIR is an intergovernmental organisation that brings together life science resources from across Europe. To coordinate the linkage of various life science efforts around modern predictive toxicology, the establishment of a new ELIXIR Community is seen as instrumental. In the past few years, joint efforts, building on incidental overlap, have been piloted in the context of ELIXIR. For example, the EU-ToxRisk, diXa, HeCaToS, transQST, and the nanotoxicology community have worked with the ELIXIR TeSS, Bioschemas, and Compute Platforms and activities. In 2018, a core group of interested parties wrote a proposal, outlining a sketch of what this new ELIXIR Toxicology Community would look like. A recent workshop (held September 30th to October 1st, 2020) extended this into an ELIXIR Toxicology roadmap and a shortlist of limited investment-high gain collaborations to give body to this new community. This Whitepaper outlines the results of these efforts and defines our vision of the ELIXIR Toxicology Community and how it complements other ELIXIR activities.

Air-Liquid Interface Exposure of Lung Epithelial Cells to Low Doses of Nanoparticles to Assess Pulmonary Adverse Effects.

Reliable and predictive in vitro assays for hazard assessments of manufactured nanomaterials (MNMs) are still limited. Specifically, exposure systems which more realistically recapitulate the physiological conditions in the lung are needed to predict pulmonary toxicity. To this end, air-liquid interface (ALI) systems have been developed in recent years which might be better suited than conventional submerged exposure assays. However, there is still a need for rigorous side-by-side comparisons of the results obtained with the two different exposure methods considering numerous parameters, such as different MNMs, cell culture models and read outs. In this study, human A549 lung epithelial cells and differentiated THP-1 macrophages were exposed under submerged conditions to two abundant types of MNMs i.e., ceria and titania nanoparticles (NPs). Membrane integrity, metabolic activity as well as pro-inflammatory responses were recorded. For comparison, A549 monocultures were also exposed at the ALI to the same MNMs. In the case of titania NPs, genotoxicity was also investigated. In general, cells were more sensitive at the ALI compared to under classical submerged conditions. Whereas ceria NPs triggered only moderate effects, titania NPs clearly initiated cytotoxicity, pro-inflammatory gene expression and genotoxicity. Interestingly, low doses of NPs deposited at the ALI were sufficient to drive adverse outcomes, as also documented in rodent experiments. Therefore, further development of ALI systems seems promising to refine, reduce or even replace acute pulmonary toxicity studies in animals.

Characterization of Nanoparticle Batch-To-Batch Variability.

A central challenge for the safe design of nanomaterials (NMs) is the inherent variability of NM properties, both as produced and as they interact with and evolve in, their surroundings. This has led to uncertainty in the literature regarding whether the biological and toxicological effects reported for NMs are related to specific NM properties themselves, or rather to the presence of impurities or physical effects such as agglomeration of particles. Thus, there is a strong need for systematic evaluation of the synthesis and processing parameters that lead to potential variability of different NM batches and the reproducible production of commonly utilized NMs. The work described here represents over three years of effort across 14 European laboratories to assess the reproducibility of nanoparticle properties produced by the same and modified synthesis routes for four of the OECD priority NMs (silica dioxide, zinc oxide, cerium dioxide and titanium dioxide) as well as amine-modified polystyrene NMs, which are frequently employed as positive controls for nanotoxicity studies. For 46 different batches of the selected NMs, all physicochemical descriptors as prioritized by the OECD have been fully characterized. The study represents the most complete assessment of NMs batch-to-batch variability performed to date and provides numerous important insights into the potential sources of variability of NMs and how these might be reduced.

Metabolic Profiling as Well as Stable Isotope Assisted Metabolic and Proteomic Analysis of RAW 264.7 Macrophages Exposed to Ship Engine Aerosol Emissions: Different Effects of Heavy Fuel Oil and Refined Diesel Fuel.

Exposure to air pollution resulting from fossil fuel combustion has been linked to multiple short-term and long term health effects. In a previous study, exposure of lung epithelial cells to engine exhaust from heavy fuel oil (HFO) and diesel fuel (DF), two of the main fuels used in marine engines, led to an increased regulation of several pathways associated with adverse cellular effects, including pro-inflammatory pathways. In addition, DF exhaust exposure was shown to have a wider response on multiple cellular regulatory levels compared to HFO emissions, suggesting a potentially higher toxicity of DF emissions over HFO. In order to further understand these effects, as well as to validate these findings in another cell line, we investigated macrophages under the same conditions as a more inflammation-relevant model. An air-liquid interface aerosol exposure system was used to provide a more biologically relevant exposure system compared to submerged experiments, with cells exposed to either the complete aerosol (particle and gas phase), or the gas phase only (with particles filtered out). Data from cytotoxicity assays were integrated with metabolomics and proteomics analyses, including stable isotope-assisted metabolomics, in order to uncover pathways affected by combustion aerosol exposure in macrophages. Through this approach, we determined differing phenotypic effects associated with the different components of aerosol. The particle phase of diluted combustion aerosols was found to induce increased cell death in macrophages, while the gas phase was found more to affect the metabolic profile. In particular, a higher cytotoxicity of DF aerosol emission was observed in relation to the HFO aerosol. Furthermore, macrophage exposure to the gas phase of HFO leads to an induction of a pro-inflammatory metabolic and proteomic phenotype. These results validate the effects found in lung epithelial cells, confirming the role of inflammation and cellular stress in the response to combustion aerosols.

Particulate matter from both heavy fuel oil and diesel fuel shipping emissions show strong biological effects on human lung cells at realistic and comparable in vitro exposure conditions.

BACKGROUND: Ship engine emissions are important with regard to lung and cardiovascular diseases especially in coastal regions worldwide. Known cellular responses to combustion particles include oxidative stress and inflammatory signalling. OBJECTIVES: To provide a molecular link between the chemical and physical characteristics of ship emission particles and the cellular responses they elicit and to identify potentially harmful fractions in shipping emission aerosols. METHODS: Through an air-liquid interface exposure system, we exposed human lung cells under realistic in vitro conditions to exhaust fumes from a ship engine running on either common heavy fuel oil (HFO) or cleaner-burning diesel fuel (DF). Advanced chemical analyses of the exhaust aerosols were combined with transcriptional, proteomic and metabolomic profiling including isotope labelling methods to characterise the lung cell responses. RESULTS: The HFO emissions contained high concentrations of toxic compounds such as metals and polycyclic aromatic hydrocarbon, and were higher in particle mass. These compounds were lower in DF emissions, which in turn had higher concentrations of elemental carbon ("soot"). Common cellular reactions included cellular stress responses and endocytosis. Reactions to HFO emissions were dominated by oxidative stress and inflammatory responses, whereas DF emissions induced generally a broader biological response than HFO emissions and affected essential cellular pathways such as energy metabolism, protein synthesis, and chromatin modification. CONCLUSIONS: Despite a lower content of known toxic compounds, combustion particles from the clean shipping fuel DF influenced several essential pathways of lung cell metabolism more strongly than particles from the unrefined fuel HFO. This might be attributable to a higher soot content in DF. Thus the role of diesel soot, which is a known carcinogen in acute air pollution-induced health effects should be further investigated. For the use of HFO and DF we recommend a reduction of carbonaceous soot in the ship emissions by implementation of filtration devices.