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The cytotoxicity of alloying elements in newly developed biodegradable metals can be assessed through relatively low-cost and rapid in vitro studies using different cell types. However, such approaches have limitations; as such, additional investigations in small mammalian models are required that recapitulate the physiological environment. In this study, we established a zebrafish (Danio rerio) model for cytotoxicity evaluations that combines the physiological aspects of an animal model with the speed and simplicity of a cell-based assay. The model was used to assess the cytotoxicity of five common alloying elements in biodegradable implant materials. Conventional in vitro testing using heart, liver, and endothelial cell lines performed in parallel with zebrafish studies revealed statistically significant differences in toxicity (up to 100-fold), along with distinct changes in the morphology of the heart, liver, and blood vessels that were undetectable in cell cultures. These results indicate that our zebrafish model is a useful alternative to mammalian systems for accurately and rapidly evaluating the in vivo toxicity of newly developed metallic materials.

Nanoparticles have resulted in great progress in biomedical imaging and targeted drug delivery in cancer theranostics. To develop nanoparticles as an effective carrier system for therapeutics, chemical structures and physicochemical properties of nanoparticle may provide a reliable means to predict the in vitro characteristics of nanoparticles. However, in vivo fates of nanoparticles, such as pharmacokinetics and tumor targeting efficiency of nanoparticles, have been difficult to predict beforehand. To predict the in vivo fates of nanoparticles in tumor-bearing mice, differences in physicochemical properties and in vitro cancer cell/macrophage uptake of 5 different nanoparticles with mean diameter of 200-250nm were comparatively analyzed, along with their circulation in adult zebrafish. The nanoparticles which showed favorable cellular uptake by macrophages indicated high unintended liver accumulation in vivo, which is attributed to the clearance by the reticuloendothelial system (RES). In addition, blood circulation of nanoparticles was closely correlated in adult zebrafish and in mice that the zebrafish experiment may elucidate the in vivo behavior of nanoparticles in advance of the in vivo experiment using mammal animal models. This comparative study on various nanoparticles was conducted to provide the basic information on predicting the in vivo fates of nanoparticles prior to the in vivo experiments.

Recent development in the field of nanomaterials has given rise into the inquiries regarding the toxicological characteristics of the nanomaterials. While many individual nanomaterials have been screened for their toxicological effects, composites that accompany nanomaterials are not common subjects to such screening through toxicological assessment. One of the widely used composites that accompany nanomaterials is catalyst composite used to reduce air pollution, which was selected as a target composite with nanomaterials for the multifaceted toxicological assessment. As existing studies did not possess any significant data regarding such catalyst composites, this study focuses on investigating toxicological characteristics of catalyst composites from various angles in both in-vitro and in-vivo settings. Initial toxicological assessment on catalyst composites was conducted using HUVECs for cell viability assays, and subsequent in-vivo assay regarding their direct influence on living organisms was done. The zebrafish embryo and its transgenic lines were used in the in-vivo assays to obtain multifaceted analytic results. Data obtained from the in-vivo assays include blood vessel formation, mutated heart morphology, and heart functionality change. Our multifaceted toxicological assessment pointed out that chemical composites augmented with nanomaterials can too have toxicological threat as much as individual nanomaterials do and alarms us with their danger. This manuscript provides a multifaceted assessment for composites augmented with nanomaterials, of which their toxicological threats have been overlooked.

Environment and organisms are persistently exposed by a mixture of various substances. However, the current evaluation method is mostly based on an individual substance's toxicity. A systematic toxicity evaluation of heterogeneous substances needs to be established. To demonstrate toxicity assessment of mixture, we chose a group of three typical ingredients in cosmetic sunscreen products that frequently enters ecosystems: benzophenone-3 (BP-3), ethylhexyl methoxycinnamate (EHMC), and titanium dioxide nanoparticle (TiO2 NP). We first determined a range of nominal toxic concentration of each ingredient or substance using Daphnia magna, and then for the subsequent organismal level phenotypic assessment, chose the wild-type zebrafish embryos. Any phenotype change, such as body deformation, led to further examinations on the specific organs of transgenic zebrafish embryos. Based on the systematic toxicity assessments of the heterogeneous substances, we offer a sequential environmental toxicity assessment protocol that starts off by utilizing Daphnia magna to determine a nominal concentration range of each substance and finishes by utilizing the zebrafish embryos to detect defects on the embryos caused by the heterogeneous substances. The protocol showed additive toxic effects of the mixtures. We propose a sequential environmental toxicity assessment protocol for the systematic toxicity screening of heterogeneous substances from Daphnia magna to zebrafish embryo in-vivo models.

Proton beam therapy can kill tumor cells while saving normal cells because of its specific energy delivery properties and so is used to various tumor patients. However, the effect of proton beam on angiogenesis in the development of blood vessels has not been determined. Here we used the zebrafish model to determine in vivo whether proton beam inhibits angiogenesis. Flk-1-GFP transgenic embryos irradiated with protons (35 MeV, spread out Bragg peak, SOBP) demonstrated a marked inhibition of embryonic growth and an altered fluorescent blood vessel development in the trunk region. When cells were stained with acridine orange to evaluate DNA damage, the number of green fluorescent cell death spots was increased in trunk regions of irradiated embryos compared to non-irradiated control embryos. Proton beam also significantly increased the cell death rate in human umbilical vein endothelial cells (HUVEC), but pretreatment with N-acetyl cystein (NAC), an antioxidant, reduced the proton-induced cell death rate (p

Lanthanide ion-doped nanophosphors are an emerging group of nanomaterials with excellent optical properties, and have been suggested as alternatives to quantum dots. In this letter, we determine the in-vitro and in-vivo toxicity of β-NaYF4:Ce,Tb nanophosphors using Capan-1 cells and embryonic zebrafish, respectively. In particular, we are the first to report on the in-vivo toxicity of β-phase nanophosphors and examine phenotypic developmental abnormalities (growth retardation, heart deformity, and bent tail), apoptotic cell death, and changes in heart function due to the nanophosphors. This study suggests the use of β-NaYF4:Ce,Tb nanophosphors as alternatives for QDs in a wide variety of biomedical imaging applications.

The present study was conducted to generate transgenic pigs coexpressing human CD55, CD59, and H-transferase (HT) using an IRES-mediated polycistronic vector. The study focused on hyperacute rejection (HAR) when considering clinical xenotransplantation as an alternative source for human organ transplants. In total, 35 transgenic cloned piglets were produced by somatic cell nuclear transfer (SCNT) and were confirmed for genomic integration of the transgenes from umbilical cord samples by PCR analysis. Eighteen swine umbilical vein endothelial cells (SUVEC) were isolated from umbilical cord veins freshly obtained from the piglets. We observed a higher expression of transgenes in the transgenic SUVEC (Tg SUVEC) compared with the human umbilical vein endothelial cells (HUVEC). Among these genes, HT and hCD59 were expressed at a higher level in the tested Tg organs compared with non-Tg control organs, but there was no difference in hCD55 expression between them. The transgenes in various organs of the Tg clones revealed organ-specific and spatial expression patterns. Using from 0 to 50% human serum solutions, we performed human complement-mediated cytolysis assays. The results showed that, overall, the Tg SUVEC tested had greater survival rates than did the non-Tg SUVEC, and the Tg SUVEC with higher HT expression levels tended to have more down-regulated α-Gal epitope expression, resulting in greater protection against cytotoxicity. By contrast, several Tg SUVEC with low CD55 expression exhibited a decreased resistance response to cytolysis. These results indicated that the levels of HT expression were inversely correlated with the levels of α-Gal epitope expression and that the combined expression of hCD55, hCD59, and HT proteins in SUVECs markedly enhances a protective response to human serum-mediated cytolysis. Taken together, these results suggest that combining a polycistronic vector system with SCNT methods provides a fast and efficient alternative for the generation of transgenic large animals with multiple genetic modifications.

We have reported that vascular endothelial growth factor (VEGF)-A induces the proliferation of human pulmonary valve endothelial cells (HPVECs) through nuclear factor in activated T cells (NFAT)c1 activation [1]. Here we show that VEGF-A increases the migration of HPVECs through NFATc1 activation, suggesting that VEGF-A/NFATc1 regulates the migration of HPVECs. To learn how this pathway may be involved in post-natal valvular repair, HPVECs were treated with VEGF-A, with or without cyclosporine A to selectively block VEGF–NFATc1 signaling. Down Syndrome critical region 1 (DSCR1) and heparin-binding EGF-like growth factor (HB-EGF) are two genes identified by DNA microarray as being up-regulated by VEGF-A in a cyclosporine-A-sensitive manner. DSCR1 silencing increased the migration of ovine valve endothelial cells, whereas HB-EGF silencing inhibited migration. This differential effect suggests that VEGF-A/NFATc1 signaling might be a crucial coordinator of endothelial cell migration in post-natal valves.