Systems toxicology of pollutant mixture released from grafted adipose tissue – CREATIvE

To address the burning question of chemical mixture effects, the CREATIvE project will take advantage of the successful pilot system developed on mice by UP. From that existing background, CREATIvE will:
- Explore the kinetics as well as the dynamics (i.e. the effects on biological systems) of an internal source of POP mixture using:
• Omics untargeted profiling technologies
• Targeted quantitative chemical measurements
• Physiologically based pharmacokinetic (PBPK) modeling
- Develop integrative systems toxicology models, based on a previously developed strategy by UP, that constitutes an interesting way of linking biological events (protein networks, complex regulations of gene clusters) to mixture of chemicals.
- Provide new data (relevant markers extrapolated to humans)

Based on this unique AT graft model, CREATIvE project will:

1. Establish a cartography of a selected POPs residues mixture in various organs and compartments to better understand their distribution among storage compartments and other target organs.
2. Decipher the impact of a POPs mixture accumulated in AT and release by complementary untargeted metabolomics (using NMR) and lipidomics (using MS) profiling.
3. Modeling of a POPs mixture using a PBPK approach, that describe the pharmacokinetic from the absorption to the elimination of the mixture in mouse. The developed PBPK model will support the integrative system biology model for the AOP development.
4. Explore the gene expression profiles and gene regulation patterns (transcriptomics) in various organs (liver, brain, AT) with bioinformatics technics to identify novel biomarkers.
Develop an integrative systems biology model to help at understanding the complex chains of events linking exposure to adverse outcome pathways (e.g. AOPs), by exploring metabolic and signaling pathways affected by the POPs (cross-omics model).

Regarding the experimental approach, a pilot study was initiated to find a common protocol for the analysis (POP titration, lipidomics and metabolomics). Based on two mice that were exposed to the POP mixture at two different doses, two naive mice were grafted with epidydimal AT from the contaminated mice. After two weeks, POP concentration were analyzed in various organs from these four mice. Four other mice were used to determine the minimal amount of tissue required for the omic analyses. The main objective being be to minimize the number of mice sacrificed, and to use the same tissues for all analysis. Three protocols were tested, and one was retained after NMR validation. Our pilot study highlights the need for a repartition of a homogenized pool of tissues for the different analysis, that possibly impacts the number of mice needed for the analysis.
A first computational model has been developed (ALTEX. 2020;37(2):287-299), based on a systems toxicology approach to explore the effects of POPs on health. A protein-protein association network (PPAN) was created using known POP-protein associations compiled from the literature and databases. This model has been used to predict the protein complexes of several candidate POPs, including perfluorooctanoic acid (PFOA). The integration of several data sources (pathways, disease annotations) involving the identified protein complexes was carried out independently to reveal putative risk factors for health. This approach has shown that several systems could be disrupted by these candidate POPs, mainly the overall metabolic system. This study highlights that in silico approach can help deciphering the putative biological mechanisms of poorly studied chemicals, and link them to possible undesirable effects in order to support regulatory assessment and trigger new epidemiological and experimental studies. In order to develop a more predictive model, the next step will be to integrate data generated by CREATIvE.

CREATIvE will provide information on the interaction of combined exposures at low doses (mixture of twelve chemicals), and their integrated effects on different biological systems. The reality of modern life is that living systems (e.g. humans or animals) are chronically exposed to a large number of chemicals, and many of them might interact using similar MoAs. Therefore, for a biologically meaningful cumulative risk assessment, approach taking into consideration not a single chemical but mixtures is required. CREATIvE considers this aspect by studying disruption of specific organs (AT, liver, brain) following exposure to a mixture of POPs.
The CREATIvE project will provide new data and tools that could be used to inform risk assessment processes performed at regulatory levels. Some of these results will also be used in parallel projects:
- the H2020- HBM4EU project (https://www.hbm4eu.eu/) such as proposal of systemic guidance values that will be elaborated on the POPs included in CREATIvE.
- the H2020 OBERON project (https://oberon-4eu.com/) to improve the reconstruction of AOP networks for endocrine disrupting chemicals.

1- Computational systems biology as an animal-free approach to characterize toxicological effects of persistent organic pollutants. Wu Q, Achebouche R, Audouze K. ALTEX. 2020;37(2):287-299. doi: 10.14573/altex.1910161.
2- Characterization of endocrine disruptors linked to metabolic related adverse outcome pathways using artificial intelligence and systems toxicology. Oral presentation- Karine Audouze. SETAC, May, 6th 2020.

Human and wildlife animals are exposed to multiple sources of environmental stressors including chemicals such as persistent organic pollutants (POPs) and endocrine disrupting compounds (EDCs). In addition to the important public health issues related to such exposures, EDCs are suspected to elicit ecosystems toxicity with an impact on the food chain and biodiversity and a significant economic burden linked to the increase of metabolic and neurodevelopmental disorders. In this complex and multifactorial context, new and innovative approaches are warranted to address potential linkages between such environmental exposure and health outcomes. Whereas exposure models in toxicology and ecotoxicology traditionally link a given external exposure source with a target organism, the vision of CREATIvE is to consider the organism as both an internal exposure source and a target. Specifically, its ambition is to assess potential health consequences from the release of POP mixtures from an internal storage site (the source) by understanding their complex biological modes of action (MoAs) on the target tissues of the same organism. It is well known that POPs bio-accumulate in living organisms and are stored in specific tissues e.g. adipose tissue (AT) brain, and liver, for long periods of time. Therefore, these tissues represent internal chronic sources of pollutants possibly leading to various disorders including metabolic and neurodegenerative diseases. Such “internal” exposures are not satisfactorily captured by current methods based on investigating different types of external POPs exposure via gavage, injection or acute inhalation. The proposed protocol will not replace the existing ones but will be complementary, taking into account for the first time internal sources of exposure.

The aim of CREATIvE is to develop a novel strategy exploring the effects of an internal exposure from grafted contaminated AT. The kinetics and consequences from a redistribution of POPs and their metabolites from grafted contaminated AT on several tissues and organs, e.g. liver, brain and host AT will be studied. The proposed integrated approach is a combination of experimental studies (chemical quantitative measurements in tissues, metabolomics, transcriptomics) and computational modeling (PBPK and systems biology approaches). The advantage of developing such integrated approaches is the possibility to identify the systemic effects of internal mixture exposure at different biological levels, by mimicking the reality of human and animal exposure. As results, new biomarkers will be characterized, and novel complementary models will be proposed which will help at increasing Agregated Exposure Pathways (AEPs) information. To our knowledge such a strategy is clearly innovative and different from existing studies. In a recent preliminary study, an allograft model was developed at Paris Descartes, consisting in a mouse graft of contaminated AT to a non-contaminated mouse. We demonstrated that four weeks after transplantation, the grafts are vascularized and functional. In those initial studies, donor AT was contaminated by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), and we showed that this contaminant was indeed redistributed to different tissues with different kinetics. Based on this acquired proof of concept, CREATIvE will explore the kinetics of a low dose POP mixture release from an internal source of exposure, and most importantly will assess the toxic effects of such mixtures on other tissues and organs. After improvement of the experimental model, a mixture of twelve environmentally relevant POPs will be studied at low doses with the aim to better understand the consequences of POP mixture release from a unique internal source of exposure.