Abstract
BackgroundIn 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.MethodsHiPSC-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.ResultsChemicals working through similar MoA (i.e., alterations of BDNF levels), at non-cytotoxic (IC20/100), very low toxic (IC5), or moderately toxic (IC20) 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.ConclusionsOur 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.
Highlights
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
Breast milk [4] and cord blood [5] have been found to contain chemicals regulated as pesticides, along with those regulated as cosmetics, and persistent organic pollutant (POP), including polychlorinated biphenyls (PCBs), confirming that simultaneous co-exposure to multiple chemicals generally occurs during pregnancy, in new-borns and toddlers
Criteria for chemical selection Based on human epidemiological data and presence of chemicals in human samples (e.g., [4, 5]), chemicals that are linked to cognitive deficit in children, including learning and memory impairment (AO of the DNT adverse outcome pathways (AOPs)) were identified and divided into two groups according to the key events (KEs) of the relevant AOPs: those were (1) impairment of brain derived neurotrophic factor (BDNF) synthesis and release associated with alterations of synaptogenesis, and (2) alteration of synaptogenesis through multiple mechanisms, not directly linked to changes of BDNF levels
Summary
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. In a study by Maffini and Neltner [2], more than 300 compounds were identified as potential DNT chemicals These chemicals belong to various regulatory silos including food contaminants, food contact materials and food additives, such as flavourings, colourings and preservatives. Taking into consideration the vulnerability of the developing brain, mixture risk assessment (MRA) for DNT effects should be implemented, since humans, including the unborn, infants and children are indisputably coexposed to more than one chemical at a time [3]. Breast milk [4] and cord blood [5] have been found to contain chemicals regulated as pesticides, along with those regulated as cosmetics (including UV filters, parabens, phthalates), and POPs, including polychlorinated biphenyls (PCBs), confirming that simultaneous co-exposure to multiple chemicals generally occurs during pregnancy, in new-borns and toddlers
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