Zebrafish High‐Throughput Screening to Study the Impact of Dissolvable Metal Oxide Nanoparticles on the Hatching Enzyme, ZHE1

Abstract

The zebrafish is emerging as a model organism for the safety assessment and hazard ranking of engineered nanomaterials. In this Communication, the implementation of a roboticized high‐throughput screening (HTS) platform with automated image analysis is demonstrated to assess the impact of dissolvable oxide nanoparticles on embryo hatching. It is further demonstrated that this hatching interference is mechanistically linked to an effect on the metalloprotease, ZHE 1, which is responsible for degradation of the chorionic membrane. The data indicate that 4 of 24 metal oxide nanoparticles (CuO, ZnO, Cr2O3, and NiO) could interfere with embryo hatching by a chelator‐sensitive mechanism that involves ligation of critical histidines in the ZHE1 center by the shed metal ions. A recombinant ZHE1 enzymatic assay is established to demonstrate that the dialysates from the same materials responsible for hatching interference also inhibit ZHE1 activity in a dose‐dependent fashion. A peptide‐based BLAST search identifies several additional aquatic species that express enzymes with homologous histidine‐based catalytic centers, suggesting that the ZHE1 mechanistic paradigm could be used to predict the toxicity of a large number of oxide nanoparticles that pose a hazard to aquatic species.

Developmental effects of two different copper oxide nanomaterials in sea urchin (Lytechinus pictus) embryos

“…In zebrafish embryos, the soluble copper released from the nano-CuO inhibits the activity of the hatching enzyme in zebrafish embryos without affecting their morphology (Lin et al, 2013). In contrast, morphology of sea urchin embryos was impacted at comparatively lower concentrations with no effect on hatching for either nano-CuO or CuSO 4 (this study); this also has been observed in Pacific herring embryos in seawater (data not shown).…”

“…Nano-CuO has other toxic effects in aquatic organisms not related to development (Gomes et al, 2011;Hanna et al, 2014;Hanna et al, 2013;Hu et al, 2014;Lin et al, 2013). In zebrafish embryos, the soluble copper released from the nano-CuO inhibits the activity of the hatching enzyme in zebrafish embryos without affecting their morphology (Lin et al, 2013).…”

“…Some studies attribute the toxic effects solely to the ions released into the media (Cho et al, 2012;Fairbairn et al, 2011;Gunawan et al, 2011;Xia et al, 2008), while others have demonstrated that NMs are more toxic than their bulk counterparts (Amorim & Scott-Fordsmand, 2012;Bielmyer-Fraser et al, 2014;Garner et al, 2015;George et al, 2012;Shi et al, 2011;Siddiqui et al, 2015;Thit et al, 2013). For example, hatching inhibition in zebrafish embryos is caused by metal oxide NM dissolution and this effect is similar to ionic forms of the metals (Lin et al, 2013;Lin et al, 2011;Xia et al, 2011), while an NM-specific effect was observed in earthworms exposed to nano-Cu (Amorim & Scott-Fordsmand, 2012). Therefore, understanding the fate, transport and transformations of NMs in different media and in different organisms during different life stages is critical to understanding their impacts in different ecosystems.…”

Acute toxicity of copper and chromium oxide nanoparticles to Daphnia similis

“…Lin et al (2012) investigated the toxicity of chromium NPs to aquatic organisms using D. rerio. This study has indicated that Cr 2 O 3 NPs could interfere with embryo hatching by a chelator-sensitive mechanism that involves ligation of critical histidine in the ZHE1 (metalloprotease, responsible for degradation of the chorionic membrane) center by the shed metal ions.…”

“…However, studies published in the international literature on the toxicity of these types of NPs to aquatic organisms are scarce. Lin et al (2012) (Knie & Lopes, 2004;Paschoalino et al, 2010). Furthermore, daphnids are considered a keystone species in aquatic toxicology because they are filter-feeders and are able to ingest NPs (Artal et al, 2013).Tests employing Daphnia have been used to evaluate the nanomaterials ecotoxicity, due to be important links in the food chain from algae consumed by them, and the fish that are predators (Paschoalino et al, 2010).They have also been proposed as a model organism for the ecotoxicological testing of nanomaterials (Paschoalino et al, 2010;Artal et al, 2013).…”