Assessment of developmental neurotoxicity induced by chemical mixtures using an adverse outcome pathway concept

Abstract

Background: In light of the vulnerability of the developing brain, mixture risk assessment (MRA) for the evaluation of developmental neurotoxicity (DNT) should be implemented, since infants and children are co-exposed to more than one chemical at a time. One possible approach to tackle MRA could be to cluster DNT chemicals in a mixture on the basis of their mode of action (MoA) into 'similar' and 'dissimilar', but still contributing to the same adverse outcome, and anchor DNT assays to common key events (CKEs) identified in DNT-specific adverse outcome pathways (AOPs). Moreover, the use of human in vitro models, such as induced pluripotent stem cell (hiPSC)derived neuronal and glial cultures would enable mechanistic understanding of chemically-induced adverse effects, avoiding species extrapolation. Methods: HiPSC-derived neural progenitors differentiated into mixed cultures of neurons and astrocytes were used to assess the effects of acute (3 days) and repeated dose (14 days) treatments with single chemicals and in mixtures belonging to different classes (i.e., lead(II) chloride and methylmercury chloride (heavy metals), chlorpyrifos (pesticide), bisphenol A (organic compound and endocrine disrupter), valproic acid (drug), and PCB138 (persistent organic pollutant and endocrine disrupter), which are associated with cognitive deficits, including learning and memory impairment in children. Selected chemicals were grouped based on their mode of action (MoA) into 'similar' and 'dissimilar' MoA compounds and their effects on synaptogenesis, neurite outgrowth, and brain derived neurotrophic factor (BDNF) protein levels, identified as CKEs in currently available AOPs relevant to DNT, were evaluated by immunocytochemistry and high content imaging analysis. Results: Chemicals working through similar MoA (i.e., alterations of BDNF levels), at non-cytotoxic (IC 20 /100), very low toxic (IC 5), or moderately toxic (IC 20) concentrations, induce DNT effects in mixtures, as shown by increased number of neurons, impairment of neurite outgrowth and synaptogenesis (the most sensitive endpoint as confirmed by mathematical modelling) and increase of BDNF levels, to a certain extent reproducing autism-like cellular changes observed in the brain of autistic children. Conclusions: Our findings suggest that the use of human iPSC-derived mixed neuronal/glial cultures applied to a battery of assays anchored to key events of an AOP network represents a valuable approach to identify mixtures of chemicals with potential to cause learning and memory impairment in children.

Establishment of an a priori protocol for the implementation and interpretation of an in‐vitro testing battery for the assessment of developmental neurotoxicity

“…set-up of test methods that already have science ready for further development. This includes for example influence of compounds on expression of BDNF and synaptogenesis (Pistollato et al, 2020). Also the differentiation of mainly GABAergic neurons was currently established (Nimtz et al, 2019) allowing assessment of GABAergic function.…”

“…Hence, the DNT testing battery covers the biological application domain of these pathways well. In addition, a recent publication highlighted the role of BDNF for many DNT-related AOPs (Pistollato 2020 The present document has been produced and adopted by the bodies identified above as author(s). This task has been carried out exclusively by the author(s) in the context of a contract between the European Food Safety Authority and the author(s), awarded following a tender procedure.…”

“…These developments envisioned for the future are shown in lavender. The synaptogenesis assay recently published (Pistollato et al 2020) will be added to the battery, personal communication Anna Price, JRC.…”

Acrylamide alters CREB and retinoic acid signalling pathways during differentiation of the human neuroblastoma SH-SY5Y cell line

“…Concomitantly with our previous study on effects of ACR after 3 and 6 days of exposure during differentiation 28 , we here found that exposure with very low (picomolar) concentrations of ACR during 9 days attenuated differentiation by sustaining proliferation and reducing neurite outgrowth. Both cell proliferation and neurite outgrowth are hallmarks of neurodevelopment and are indicated as endpoints to be included in strategies for assessment of DNT using in vitro approaches 37 . Ogawa et al 38 showed that low doses of ACR in drinking water during gestation and weaning period decreased apoptosis of neuroblasts in hippocampus, suggesting a compensatory mechanism for impaired neurogenesis 38 .…”

“…A cell model like the SH-SY5Y cell line, which is easy to handle, robust and relatively fast to differentiate, is an attractive tool for such studies, e.g. as a cheaper and less sophisticated alternative to human inducible pluripotent stem cell (hiPSC) models 37 . However, our results showing that concentrations of ACR that are relevant for human exposure may be harmful for developing neurons, should be validated in other, more complex models of the nervous system.…”