Background: The disorder of copper homeostasis is linked with disease and developmental defects, and excess copper_nanoparticles (CuNPs) and ion (Cu²⁺) will induce developmental malformation and disease in organisms. However, little knowledge is available regarding its potential regulation mechanisms, and little study links excess copper with retinal developmental malformation and disease.Methods: Embryos were stressed with copper (CuNPs and Cu²⁺), and cell proliferation and apoptosis assays, reactive oxygen species (ROS) and endoplasmic reticulum (ER) signaling detections, and genetic mutantscox17^−/−andatp7a^−/−application, were used to evaluate copper induced retinal developmental malformation and the underlying genetic and biological regulating mechanisms.Results: Copper reduced retinal cells and down-regulated expression of retinal genes, damaged the structures of ER and mitochondria in retinal cells, up-regulated unfold protein responses (UPR) and ROS, and increased apoptosis in copper-stressed retinal cells. The copper induced retinal defects could be significantly neutralized by ROS scavengers reduced Glutathione (GSH) & N-acetylcysteine (NAC) and ER stress inhibitor 4- phenylbutyric acid (PBA). Blocking the transportation of copper to mitochondria, or to trans-Golgi network and to be exported into plasma, by deleting genecox17oratp7a, could alleviate retinal developmental defects in embryos under copper stresses.Conclusions: This is probably the first report to reveal that copper nanoparticles and ions induce retinal developmental defects via upregulating UPR and ROS, leading to apoptosis in zebrafish embryonic retinal cells. Integrated function of copper transporter (Cox17 and Atp7a) is necessary for copper induced retinal defects.Graphical

Copper-induced delayed hatching and dysfunctional movement had been reported previously, and unbalanced free copper was found in the body of humans with Alzheimer’s disease and other neural diseases, but details of the underlying mechanisms are still unknown. In this study, zebrafish (Danio rerio) embryos exposed to over 3.9μM of copper exhibited delayed hatching and significantly dysfunctional movement. Using high-throughput in situ hybridization screening and by conducting an in-depth analysis of gene characterization in embryos exposed to copper, we found that copper caused neural crest defects from the initiation stage of neurogenesis, and embryos younger than the 70% epiboly stage were sensitive to copper toxicity. The myelination of Schwann cells, other than melanophores, cartilage, and neurons, was inhibited by copper during neurogenesis. In addition, axon guidance was blocked by copper. Down-regulated cdx4-hox might have contributed to the neurogenesis-related defects. Moreover, copper inhibited the differentiation of muscle fibers and myotomes but not the specification of muscle progenitors. In summary, our data reveal a novel molecular mechanism for copper-inhibited locomotor behavior in embryos, in which copper blocks functional muscle fiber specification during myogenesis and inhibits the specification of axons and Schwann cell myelination during neurogenesis. A combination of these processes results in dysfunctional locomotor behavior in zebrafish embryos exposed to copper.

As an essential trace element, copper plays key roles in the activation of multiple enzymes, neurotransmitter biosynthesis and denaturation, as well as the decomposition of superoxide and the synthesis of collagen. The intestines is the main organ for copper absorption and transfer, and intestinal copper accumulation is observed in some patients with gene mutations. However, a vertebrate model to link copper accumulation with intestinal diseases and defects is still lacking, and the data concerning the mechanisms underlying this link are still scarce. In this study, the effects of exogenous copper (CuNPs or their released Cu²⁺) on intestinal development in zebrafish embryos were investigated after their exposure to copper. The results showed that over 0.10 mg L⁻¹ of CuNPs or Cu²⁺ damaged the zebrafish intestinal development, including thinned epithelial cells as well as few and shortened intestinal villi. Under CuNP stress, the expression was significantly (p < 0.05) reduced for the intestinal marker genes (slc15a1b, cyp3a65, cyp8b1, fabp2), but increased for the endoplasmic reticulum (ER) stress marker (bip) in the zebrafish intestines. Additionally, immunofluorescence analysis revealed that CuNPs or Cu²⁺ induced the production of ER stress (indicated by PDI) and oxidative stress (indicated by 4-HNE) in the intestinal cells. The expression of the aforementioned intestinal marker genes could be restored to normal by inhibiting the production of ER stress or oxidative stress with ER stress alleviator PBA (4-phenylbutyric acid) or ROS scavengers GSH (reduced Glutathione) or NAC (Nacetylcysteine) in CuNP or Cu²⁺ stressed embryos, suggesting that copper induces intestinal defects mainly by ER and oxidative stress. Moreover, obvious intestinal defects were observed in copper-stressed cox17^−/− and atp7a^−/− mutants, implying that blocking the transportation of copper to the mitochondria or trans-Golgi network by deleting cox17 or atp7a could not alleviate copper-induced intestinal developmental defects. This is probably the first report to reveal that copper nanoparticles (CuNPs) and their released ions (Cu²⁺) cause intestinal developmental defects via inducing ER and ROS stresses. It is also the first report on the intestinal developmental responses of cox17^−/− or atp7a^−/− mutants to copper stimulation.