Département Toxicologie de l’environnement

Humpback whales, like other polar wildlife, accumulate persistent organic pollutants. In Southern hemisphere populations, hexachlorobenzene (HCB) dominates the contaminant profiles. HCB is linked to a variety of health effects and is classified as a group 2B carcinogen, but the mechanism of action is a matter of contention. Potential toxicological effects to humpback whales remain entirely unknown. The recently established humpback whale fibroblast cell line (HuWa) offers an in vitro model for toxicological investigations. We here combine this novel cell line with a passive dosing strategy to investigate whale-specific toxicity of HCB. The relevant partitioning coefficients were determined to produce stable and predictable exposure concentrations in small-scale bioassays. The system was used to assess acute toxicity as well as genotoxicity of HCB to the HuWa cell line. While we found some transient reductions in metabolic activity, measured with the indicator dye alamarBlue, no clear acute toxic effects were discernible. Yet, a significant increase in DNA damage, detected in the alkaline comet assay, was found in HuWa cells exposed to 10 μg L^-1 HCB during the sensitive phase of cell attachment. Collectively, this work provides a ready-to-use passive dosing system and delivers evidence that HCB elicits genotoxicity in humpback whale cells.

1-Chloro-2,4-dinitrobenzene (CDNB) is widely used as a model substrate for measuring enzyme activity of glutathione S-transferases in toxicity studies and in studies focusing on the metabol-ic capacity of different test systems. To allow the quantification of CDNB at low, non-toxic concentra-tions, we developed a sensitive liquid chromatography-mass spectrometry (LC-MS) technique, which is based on electron capture ionization using atmospheric pressure chemical ionization (APCI) in negative ion mode. Gas phase reactions occurring under atmospheric pressure produce specific ions that allow direct CDNB quantification down to 17 ng/ml in water. Using the new technique, we were able to verify CDNB exposure concentrations applied in two typical toxicity studies with early life stages of the com-mon model organisms, zebrafish (Danio rerio) and a zebrafish embryonic cell line (Pac2).

The use of omics is gaining importance in the field of nanoecotoxicology; an increasing number of studies are aiming to investigate the effects and modes of action of engineered nanomaterials (ENMs) in this way. However, a systematic synthesis of the outcome of such studies regarding common responses and toxicity pathways is currently lacking. We developed an R-scripted computational pipeline to perform reanalysis and functional analysis of relevant transcriptomic data sets using a common approach, independent from the ENM type, and across different organisms, including Arabidopsis thaliana, Caenorhabditis elegans, and Danio rerio. Using the pipeline that can semiautomatically process data from different microarray technologies, we were able to determine the most common molecular mechanisms of nanotoxicity across extremely variable data sets. As expected, we found known mechanisms, such as interference with energy generation, oxidative stress, disruption of DNA synthesis, and activation of DNA-repair but also discovered that some less-described molecular responses to ENMs, such as DNA/RNA methylation, protein folding, and interference with neurological functions, are present across the different studies. Results were visualized in radar charts to assess toxicological response patterns allowing the comparison of different organisms and ENM types. This can be helpful to retrieve ENM-related hazard information and thus fill knowledge gaps in a comprehensive way in regard to the molecular underpinnings and mechanistic understanding of nanotoxicity.

The analysis of larval zebrafish locomotor behavior has emerged as a powerful indicator of perturbations in the nervous system and is used in many fields of research, including neuroscience, toxicology and drug discovery. The behavior of larval zebrafish however, is highly variable, resulting in the use of large numbers of animals and the inability to detect small effects. In this study, we analyzed whether individual locomotor behavior is stable over development and whether behavioral parameters correlate with physiological and morphological features, with the aim of better understanding the variability and predictability of larval locomotor behavior. Our results reveal that locomotor activity of an individual larva remains consistent throughout a given day and is predictable throughout larval development, especially during dark phases, under which larvae demonstrate light-searching behaviors and increased activity. The larvae’s response to startle-stimuli was found to be unpredictable, with no correlation found between response strength and locomotor activity. Furthermore, locomotor activity was not associated with physiological or morphological features of a larva (resting heart rate, body length, size of the swim bladder). Overall, our findings highlight the areas of intra-individual consistency, which could be used to improve the sensitivity of assays using zebrafish locomotor activity as an endpoint.

In this study we present the first fish-gut-on-chip model. This model is based on the reconstruction of the intestinal barrier by culturing two intestinal cell lines from rainbow trout, namely epithelial RTgutGC and fibroblastic RTgutF, in an artificial microenvironment. For a realistic mimicry of the interface between the intestinal lumen and the interior of the organism we i) developed ultrathin and highly porous silicon nitride membranes that serve as basement membrane analogues and provide a culture interface for the fish cells; ii) constructed a unique micro-well plate-based microfluidic bioreactor that enables parallelization of experiments and creates realistic fluid flow exposure scenarios for the cells; iii) integrated electrodes in the reactor for non-invasive impedance sensing of cellular well-being. In a first approach, we used this reactor to investigate the response of epithelial fish cells to in vivo-like shear stress rates of 0.002–0.06 dyne per cm², resulting from fluid flow within the intestinal lumen. Moreover, we investigated the interplay of epithelial and fibroblast cells under optimal flow conditions to carefully evaluate the benefits and drawbacks of the more complex reconstruction of the intestinal architecture. With our fish-gut-on-chip model we open up new strategies for a better understanding of basic fish physiology, for the refinement of fish feed in aquaculture and for predicting chemical uptake and bioaccumulation in fish for environmental risk assessment. The basic principles of our reactor prototype, including the use of ultrathin membranes, an open microfluidic circuit for perfusion and the micro-well plate-based format for simplified handling and avoidance of air-bubbles, will as well be of great value for other barrier-on-chip models.

Titanium dioxide nanoparticles (TiO₂ NPs) are widely incorporated in various consumer products such as cosmetics and food. Despite known human exposure, the potential risks of TiO₂ NPs during pregnancy are not fully understood, but several studies in mice elucidated toxic effects on fetal development. It has also been shown that modifying NPs with positive or negative surface charge alters cellular uptake and abolishes fetotoxicity of silicon dioxide (SiO₂) NPs in mice.
Here, we investigated accumulation and translocation of positively charged TiO₂-NH₂ and negatively charged TiO₂-COOH NPs at the placental barrier, to clarify whether surface charge provides a means to control TiO₂ NP distribution at the placental barrier. To ensure outcome relevant for humans, the recently developed in vitro human placental co-culture model and the gold standard amongst placental translocation models – the ex vivo perfusion of human term placental tissue - were employed during this study. Sector field-ICP-MS analysis of maternal and fetal supernatants as well as placental cells/tissues revealed a substantial accumulation of both TiO₂ NP types while no considerable placental translocation was apparent in both models. Characterization of agglomeration behavior demonstrated a strong and fast agglomeration of TiO₂-NH₂ and TiO₂-COOH NPs in the different culture media.
Overall, our results indicate that surface charge is not a key factor to steer placental uptake and transfer of TiO₂. Moreover, the negligible placental transfer but high accumulation of TiO₂ NPs in placental tissue suggests that potential effects on fetal health may occur indirectly, which calls for further studies elucidating the impact of TiO₂ NPs on placental tissue functionality and signaling.