Work Package 4 - AeroTOX

In WP4 (AeroTOX), efficient read-out of the Aerosol induced mechanisms of biological and TOXicological effects induced by different types of aerosol (SOA, biomass burning, and dust) with advanced exposure models will be developed, optimized and applied. Crucial for predicting the effects of ambient aerosol health effects is the use of comprehensive set of cell cultures, silenced cell lines, co-cultures, tissue cultures and in vivo experiments accompanied by a state-of-the-art molecular biological and toxicological effect analyses. By developing lung cell culture models to co-cultures with cells from other organs, the covered effects will be extended beyond the lung and enable a better prediction of human health effects. The partners WIS and HMGU contribute to this WP. The cell and tissue culture-based results will be tested and validated with careful use of in vivo models, thus adhering to the guiding principle for ethical use of animal testing (3R principle: reduce, replace, refine).
Large Facilities of the Helmholtz International Laboratory: WP4 will take use of the animal facilities, the core omics characterization and biological labs at WIS as well as the toxicological and biological laboratories at HMGU. New infrastructure such as advanced electron microscopies and imaging of metabolites and cell features will be used by all three partners as they become available in WIS.


The objectives of WP 4 are to get a deeper insight in the biological effects of aging and atmospherically transforming of anthropogenic and biogenic aerosols. In particular the question, whether a toxification of anthropogenic emissions upon aging in the atmosphere with respect to acute cytotoxicity as well as genotoxic and epigenetic effects is observed, or not, is considered. Therefore, W4 will develop and apply disease relevant, sensitive and validated in vitro lung cell and tissue models as well as suited animal models for validation of the observed effects.

Model typeModelsBiological characterization endpoints
Cell CulturesLung (A549, BEAS2B, and alveolar
macrophages NR8383), Liver (AML-
12, mouse), Cardiocytes, Silenced
cells for specific key genes (shRNA
Toxicity: MTT, LDH, ROS, Oxidative damage, evaluation of cell death
mechanisms by flow cytometry, Gene expression: Microarray and
bioinformatics analysis (facilities will be provided at the WIS using Affymetrix
MAS5), Mitochondria: Seahorse analyzer, confocal microscopy for
mitochondrial potential
3D-TissuesMucilAir™ Human Airway Epithelia
consisting of primary epithelial cells ,
MucilAir™-HF Human Airway Epithelia
consisting of primary epithelial cells
co-cultured with Human airway
Fibroblasts (HF), 3D MucilAir
containing specific diseases
Gene expression: Microarray and bioinformatics analysis, facilities provided
at WIS using Affymetrix MAS5., Inflammation: Cytokine secretion, Protein
profiling: targeted proteomics, facilities will be provided at WIS, Histology
and morphology (facilities provided WIS)., Oxidative damage: adducts of
lipid and protein damage Epigenetic changes: DNA methylation
In vivo exposureMice exposure for validation and
mechanistic understanding (to highly
impact aerosols, following RRR
Systemic inflammatory evaluation: Blood, broncho-alveolar lung fluid
(BALF) by image stream flow cytometry for specific inflammatory markers,
Gene expression: Microarray and bioinformatics analysis (facilities: WIS)
using Affymetrix MAS5), Oxidative damage: adducts of lipid and protein
damage, Epigenetic changes: DNA methylation
Histology and morphology: (facilities will be provided at WIS)
=> All in vivo experiment will be approved by the Weizmann
 Institute Animal Care and Use Committees (IACUC)

By targeting the toxicological/omics methods as well as developing simpler biological assays, a routine toxicological monitoring of air pollution is a long-term goal. The respiratory and oral tracts along with the skin are the common routes by which humans are exposed to a wide variety of ambient pollutants. Therefore, we will study effects of exposure on human lung, liver (represents secondary organ of exposure) and skin (epidermis) cells. Subsequent to the exposure (Air-Liquid Interface (ALI) or off-line exposure using extracts), the human cell cultures and in vivo models will be subjected to an innovative and refined molecular biological analysis for highly sensitive, high throughput and comprehensive detection of adverse cellular and whole tissue responses. State of the science cultures and 3D tissue models will be used:

i) adaptation of the cell culturing and exposure protocols representing different tissues,

ii) development of cell co-culture models and usage of lung 3D tissues for better description of disease-specific end-points,

iii) cells silenced for specific mechanisms, such as the Nrf2-related protection pathways (see Box below), and

iv) in vivo exposure using mice for systemic evaluation of aerosol exposure.

By using macrophage cultures, a better description of inflammatory responses for long-term exposures is envisaged. For specific endpoints, in particular beyond the lung, existing co-culture expertise is used for setting up disease-directed co-culture models using lung cells (alveolar or bronchial epithelial cells and/or macrophages for inflammatory endpoints) in conjunction with i) hepatocytes (metabolic activation), ii) fibroblasts (fibrosis induction), iii) neuronal cells (effects on neuronal system), iv) cardiac pacemaker cells (influence of the cardiovascular system). 3D tissue models enable a more realistic description of inflammatory and molecular responses enduring long-term exposure. Different endpoints (e.g. secretion of specific interleukins, cell viability and integrity, gene screen analysis, mitochondrial function, tissue histology, and advanced imaging) will be performed. For specific endpoints (such as mitochondrial function, metabolism and cell imaging), the seahorse analyzer and state of the art confocal microscopy with specific “reporters” will be used. Gene microarray and advanced bioinformatics will be used, in addition to gene specific function using silenced gene techniques (shRNA) for cell cultures. This will provide the basis for the deep molecular biological analyses required to cope with the low concentrations of ambient aerosols. The ALI exposure systems will be coupled to several setups that will allow controlled aerosol aging (WP2) and comprehensive chemical analysis of the aerosol composition (WP3). The study will provide comprehensive characterization of biological mechanisms of toxicity relative to health risk with regard to the respective emission sources and their aging mechanisms.