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.

Dietary boron intake is associated with reduced prostate and lung cancer risk and increased bone mass. Boron is absorbed and circulated as boric acid (BA) and at physiological concentrations is a reversible competitive inhibitor of cyclic ADP ribose, the endogenous agonist of the ryanodine receptor calcium (Ca+2) channel, and lowers endoplasmic reticulum (ER) [Ca2+]. Low ER [Ca2+] has been reported to induce ER stress and activate the eIF2α/ATF4 pathway. Here we report that treatment of DU-145 prostate cells with physiological levels of BA induces ER stress with the formation of stress granules and mild activation of eIF2α, GRP78/BiP, and ATF4. Mild activation of eIF2α and its downstream transcription factor, ATF4, enables cells to reconfigure gene expression to manage stress conditions and mild activation of ATF4 is also required for the differentiation of osteoblast cells. Our results using physiological levels of boric acid identify the eIF2α/ATF pathway as a plausible mode of action that underpins the reported health effects of dietary boron.

Boron is abundant in vegetables, nuts, legumes, and fruit and intake is associated with reduced risk of cancer and DNA damage and increased antioxidant status. Blood boric acid (BA) levels are approximately 10 μM BA in men at the mean US boron intake. Treatment of DU-145 human prostate cancer cells with 10 μM BA stimulates phosphorylation of elongation initiation factor 2α (eIF2α) at Ser51 leading to activation of the eIF2α/ATF4 pathway which activates the DNA damage-inducible protein GADD34. In the present study, we used MEF WT and MEF PERK (±) cells to test the hypothesis that BA-activated eIF2α phosphorylation requires protein kinase RNA-like endoplasmic reticulum kinase (PERK) and activates Nrf2 and the antioxidant response element (ARE). BA (10 μM) increased phosphorylation of eIF2α Ser51 in MEF WT cells at 1 h, but not in MEF Perk -/- cells exposed for as long as 6 h. GCN2 kinase-dependent phosphorylation of eIF2α Ser51 was activated in MEF PERK -/- cells by amino acid starvation. Nrf2 phosphorylation is PERK dependent and when activated is translocated from the cytoplasm to the nucleus where it acts as a transcription factor for ARE. DU-145 cells were treated with 10 μM BA and Nrf2 measured by immunofluorescence. Cytoplasmic Nrf2 was translocated to the nucleus at 1.5-2 h in DU-145 and MEF WT cells, but not MEF PERK -/- cells. Real-time PCR was used to measure mRNA levels of three ARE genes (HMOX-1, NQO1, and GCLC). Treatment with 10 μM BA increased the mRNA levels of all three genes at 1-4 h in DU-145 cells and HMOX1 and GCLC in MEF WT cells. These results extend the known boric acid signaling pathway to ARE-regulated genes. The BA signaling pathway can be expressed using the schematic [BA + cADPR → cADPR-BA → [[ER] Ca↓] → 3 pathways: PERK/eIF2αP → pathways ATF4 and Nrf2; and [[ER] Ca↓] → ER stress → ATF6 pathway. This signaling pathway provides a framework that links many of the molecular changes that underpin the biological effects of boron intake.

Fruits, nuts, legumes, and vegetables are rich sources of boron (B), an essential plant nutrient with chemopreventive properties. Blood boric acid (BA) levels reflect recent B intake, and men at the US mean intake have a reported non-fasting level of 10 μM. Treatment of DU-145 prostate cancer cells with physiological concentrations of BA inhibits cell proliferation without causing apoptosis and activates eukaryotic initiation factor 2 (eIF2α). EIF2α induces cell differentiation and protects cells by redirecting gene expression to manage endoplasmic reticulum stress. Our objective was to determine the temporal expression of endoplasmic reticulum (ER) stress-activated genes in DU-145 prostate cells treated with 10 μM BA. Immunoblots showed post-treatment increases in eIF2α protein at 30 min and ATF4 and ATF6 proteins at 1 h and 30 min, respectively. The increase in ATF4 was accompanied by an increase in the expression of its downstream genes growth arrest and DNA damage-induced protein 34 (GADD34) and homocysteine-induced ER protein (Herp), but a decrease in GADD153/CCAAT/enhancer-binding protein homologous protein (CHOP), a pro-apoptotic gene. The increase in ATF6 was accompanied by an increase in expression of its downstream genes GRP78/BiP, calreticulin, Grp94, and EDEM. BA did not activate IRE1 or induce cleavage of XBP1 mRNA, a target of IRE1. Low boron status has been associated with increased cancer risk, low bone mineralization, and retinal degeneration. ATF4 and BiP/GRP78 function in osteogenesis and bone remodeling, calreticulin is required for tumor suppressor p53 function and mineralization of teeth, and BiP/GRP78 and EDEM prevent the aggregation of misfolded opsins which leads to retinal degeneration. The identification of BA-activated genes that regulate its phenotypic effects provides a molecular underpinning for boron nutrition and biology.Electronic supplementary materialThe online version of this article (doi:10.1007/s12011-016-0824-y) contains supplementary material, which is available to authorized users.

Zebrafish is emerging as a model organism for the safety assessment and hazard ranking of engineered nanomaterials. , A. E. Nel and co‐workers showcase a highly automated high‐throughput screening (HTS) platform using zebrafish embryos for the hazard assessment of 24 representative metal oxide nanoparticles. Through HTS analysis, four metal oxide nanoparticles are found to interfere with zebrafish embryo hatching. It is further demonstrated that hatching interference is a result of toxic metal ion shed from nanoparticles, compromising the zebrafish hatching enzyme 1 (ZHE1) activity. The structural and functional similarities of hatching enzymes across fish species suggest that the ZHE1 mechanistic paradigm could be used to predict the toxicity of a large number of engineered nanoparticles that may be hazardous to aquatic species.