Sten Linnarsson of the Karolinska Institutet kicked off the first session of the second day with a talk on the molecular anatomy of the mouse brain. The brain is arguably the most complex organ in all biology. To understand how it works, you must first understand how it is built; what are the components, how are they connected, and how do they talk to each other? With the recent development of single-cell RNA sequencing, it has become increasingly feasible to map the cell types of the brain without the use of predefined markers. Recently, Linnarsson and his colleagues applied this strategy to the mouse cortex and hippocampus and discovered a total of 47 cell types, including novel types of neurons, glia and vascular cells. Generalising this strategy, it will be possible to create a complete molecular anatomy of the mouse brain – that is, a complete and detailed inventory of the parts from which a brain is built.
Next up was Brian D Thrall, who spoke about his work studying the effects of ENMs on macrophages using integrated ‘omics. The vast diversity of ENMs under development is driving demand for new approaches for predictive toxicological and hazard analysis, placing increased importance on in vitro cellular test systems for nanotoxicology that can be conducted with rapid throughput. Growing epidemiological evidence links exposure to nanoscale particulates from ambient and occupational sources with increased morbidity and susceptibility to lung infections, including pneumonia. The team have shown that macrophages use common scavenger receptor-mediated pathways to recognise and internalise both ENMs and bacterial pathogens. Whole genome transcriptomics studies reveal that following receptor-mediated uptake of some ENMs, macrophages display dysregulated patterns of gene activation, hallmarked by exaggerated activation of antioxidant response pathways and suppressed activation of both pro and anti-inflammatory pathways. This results in reduced phagocytic capacity towards Streptococcus pneumonia. Guided by these findings, the team has developed high throughput in vitro functional assays to assess the dose-depndent effects of more than 20 different types of ENM pre-treatment on the phagosomal functions of macrophages towards S. pneumonia. Parallel studies in vivo demonstrated that exposure of mice to selected ENMs, including materials which show no cytoxic or pro-inflammatory activity alone, inhibited bacterial clearance and exacerbated lung infections caused by S. pneumonia. Furthermore, these in vivo outcomes were accurately predicted by in vitro assays of macrophage phagosomal function. QSAR modelling grounded with cell dosimetry analysis predicts that the potency by which metal oxide particles inhibit macrophage phagocytosis is related to the ease with which they participate in electron transfer reactions and promote cellular redox stress. The team has tested this hypothesis at the molecular level using a novel proteomic strategy that permits global and site-specific identification of redox modifications to proteins. This has allowed the team to quantify, for the first time at a proteome-wide scale, the specific intracellular protein targets and pathways susceptible to redox modifications induced by ENM exposure. Analyses show that different protein pathways are susceptible to oxidative modification following exposure to ENMs that induce low versus high redox stress, with proteins associated with regulation of phagocytosis, protein translation and protein stability among the most sensitive macrophage pathways identified. This integrated ‘omics-based approach has provided unique insights into the specific intracellular redox targets and gene regulatory pathways that underlie altered macrophage innate immune function in response to ENM exposure. Furthermore, the results illustrate the important concept that adverse biological consequences of nanomaterials may not always be directly mediated, but can be manifested by altering susceptibility to other common environmental exposures.
Mark Viant of the University of Birmingham finished the session with a talk on the effects of zinc oxide nanoparticles. Zinc oxide particles are one of the most widely used nanomaterials, applied in consumer products from cosmetics to electronics. Nevertheless, our understanding of their aquatic toxicity is limited, although it is speculated to arise from dissolution of Zn2+ from the nanoparticles. Metabolomics, the study of the small molecule composition of biological samples, is a proven approach for discovering metabolic responses to toxicants. Viant’s group exploited the non-targeted capabilities of metabolomics to investigate the molecular toxicity of waterborne ZnO nanoparticles to Daphnia magna. Mass spectrometry based metabolomics showed a dose dependent metabolic response to the nanoparticles, orthogonal to the effects of bulk ZnO and Zn2+. Detailed investigations revealed that families of aliphatic sulphates and sulphamates were significantly decreased following ZnO nanoparticle exposure. Some of these compounds have previously been shown to act as kairomones; chemical messengers emitted by the Daphnia that benefit another species without benefitting the emitter. These kairomones released by the daphniids are sensed by some algal species, which subsequently alter their morphology in direct response to this predation threat. Using the unicellular alga Desmodesmus subspicatus, the group has replicated this morphological response to the kairomones of healthy D. magna, and more importantly haa shown a modulation of this algal morphological response following exposure of D. magna to ZnO nanoparticles. In addition, they have used several imaging modalities to reveal the uptake of ZnO nanoparticles into the Daphnia gut and associated cellular perturbations that underpin this toxicity. Collectively these discoveries represent a novel nanoparticle-biota interaction with currently uncharacterised ecological consequences.
Mónica Amorim of the University of Aveiro gave a short talk on the new challenges presented by nanomaterials to the standard effect assessment. One way forward could be to link sub organismal high-throughput based responses to population outcomes, because effects on organisms are preceded by earlier changes at the sub organismal level (cells, genes). This will allow for rapid detection of effects during shorter exposure time and an understanding of the mechanisms of toxicity, towards a systems toxicology approach. Moreover, given the link, such data can be integrated onto Adverse Outcome Pathways (AOPs) and support a knowledge based risk assessment. Finally, this approach also allows for detection of population effect in field organisms.