In Vivo Detection of Mitochondrial Dysfunction Induced by Clinical Drugs and Disease-Associated Genes Using a Novel Dye ZMJ214 in Zebrafish.

Mitochondrial dysfunction has been implicated in various drug-induced toxicities and genetic disorders. Recently, the zebrafish has emerged as a versatile animal model for both chemical and genetic screenings. Taking advantage of its transparency, various in vivo fluorescent imaging methods have been developed to identify novel functions of chemicals and genes in zebrafish. However, there have not been fluorescent probes that can detect mitochondrial membrane potential in living zebrafish. In this study, we identified a novel cyanine dye called ZMJ214 that detects mitochondrial membrane potential in living zebrafish from 4 to 8 days post fertilization and is administered by simple immersion. The fluorescence intensity of ZMJ214 in zebrafish was increased and decreased by oligomycin and FCCP, respectively, suggesting a positive correlation between ZMJ214 fluorescence and mitochondrial membrane potential. In vivo imaging of zebrafish stained with ZMJ214 allowed for the detection of altered mitochondrial membrane potential induced by the antidiabetic drug troglitazone and the antiepileptic drug tolcapone, both of which have been withdrawn from the market due to mitochondrial toxicity. In contrast, pioglitazone and entacapone, which are similar to troglitazone and tolcapone, respectively, and have been used commercially, did not cause a change in mitochondrial membrane potential in zebrafish stained with ZMJ214. Live imaging of zebrafish stained with ZMJ214 also revealed that knock-down of slc25a12, a mitochondrial carrier protein associated with autism, dysregulated the mitochondrial membrane potential. These results suggest that ZMJ214 can be a useful tool to identify chemicals and genes that cause mitochondrial dysfunction in vivo.

In vivo selective imaging and inhibition of leukemia stem-like cells using the fluorescent carbocyanine derivative, DiOC5(3).

Elimination of leukemia stem cells (LSCs) is necessary for the destruction of malignant cell populations. Owing to the very small number of LSCs in leukemia cells, xenotransplantation studies are difficult in terms of functionally and pathophysiologically replicating clinical conditions of cell culture experiments. There is currently a limited number of lead compounds that target LSCs. Using the LSC-xenograft zebrafish screening method we previously developed, we found that the fluorescent compound 3,3'-dipentyloxacarbocyanine iodide (DiOC5(3)) selectively marked LSCs and suppressed their proliferation in vivo and in vitro. DiOC5(3) had no obvious toxicity to human umbilical cord blood CD34+ progenitor cells and normal zebrafish. It accumulated in mitochondria through organic anion transporter polypeptides that are overexpressed in the plasma membrane of LSCs, and induced apoptosis via ROS overproduction. DiOC5(3) also inhibited the nuclear translocation of NF-κB through the downregulation of LSC-selective pathways, as indicated from DNA microarray analysis. In summary, DiOC5(3) is a new type of anti-LSC compound available for diagnostic imaging and therapeutics that has the advantage of being a single fluorescent chemical.

Zebrafish xenotransplantation model for cancer stem-like cell study and high-throughput screening of inhibitors.

Xenotransplantation studies are important tools for studying cancer biology, especially for assaying tumor cell malignancy and providing cancer information in vivo. Cancer stem-like cells (CSCs) have been identified in many cancer types to drive tumor growth and recurrence, from "keeping" to "keep" resistant to chemotherapy and radiation therapy. In this study, we developed the xenotransplantation of CSCs derived from the leukemia and solid tumor cell lines using the zebrafish models. In adult zebrafish, we investigated that the xenografted leukemia stem cells (LSCs) from K562 cells could proliferate in vivo and keep the cancer property by re-transplantation. As for the solid tumor, these CSCs from DU145 cells (human prostate cancer) and HepG2 cells (human liver cancer) could form the tumor mass and even metastasis after xenotransplantation. In addition, the zebrafish embryos with CSC xenotransplantation could evaluate docetaxel in vivo efficiently and be available to screen the novel inhibitors by high-throughput manner. In summary, these zebrafish xenotransplantation models devote a good platform for the CSC mechanism investigation and anti-CSC inhibitor screening.

Identification of a novel indoline derivative for in vivo fluorescent imaging of blood-brain barrier disruption in animal models.

Disruption of the blood-brain barrier (BBB) can occur in various pathophysiological conditions. Administration of extraneous tracers that can pass the disrupted, but not the intact, BBB and detection of the extravasation have been widely used to assess BBB disruption in animal models. Although several fluorescent tracers have been successfully used, the administration of these tracers basically requires intravascular injection, which can be laborious when using small animals such as zebrafish. To identify fluorescent tracers that could be easily administered into various animal models and visualize the BBB disruption in vivo, we prepared nine structurally related indoline derivatives (IDs) as a minimum set of diverse fluorescent compounds. We found that one ID, ZMB741, had the highest affinity for serum albumin and emitted the strongest fluorescence in the presence of serum albumin of the nine IDs tested. The affinity to serum albumin and the fluorescence intensity was superior to those of Evans blue and indocyanine green that have been conventionally used to assess the BBB disruption. We showed that ZMB741 could be administered into zebrafish by static immersion or mice by intraperitoneal injection and visualizes the active disruption of their BBB. These results suggest that ZMB741 can be a convenient and versatile tool for in vivo fluorescent imaging of BBB disruption in various animal models. The strategy used in this study can also be applied to diversity-oriented libraries to identify novel fluorescent tracers that may be superior to ZMB741.

The use of fluorescent indoline dyes for side population analysis.

Dye efflux assay evaluated by flow cytometry is useful for stem cell studies. The side population (SP) cells, characterized by the capacity to efflux Hoechst 33342 dye, have been shown to be enriched for hematopoietic stem cells (HSCs) in bone marrow. In addition, SP cells are isolated from various tissues and cell lines, and are also potential candidates for cancer stem cells. However, ultra violet (UV) light, which is not common for every flow cytometer, is required to excite Hoechst 33342. Here we showed that a fluorescent indoline dye ZMB793 can be excited by 488-nm laser, equipped in almost all the modern flow cytometers, and ZMB793-excluding cells showed SP phenotype. HSCs were exclusively enriched in the ZMB793-excluding cells, while ZMB793 was localized in cytosol of bone marrow lineage cells. The efflux of ZMB793 dye was mediated by ATP binding cassette (ABC) transporter Abcg2. Moreover, staining properties were affected by the side-chain structure of the dyes. These data indicate that the fluorescent dye ZMB793 could be used for the SP cell analysis.

In vivo assessment of the permeability of the blood-brain barrier and blood-retinal barrier to fluorescent indoline derivatives in zebrafish.

BACKGROUND: Successful delivery of compounds to the brain and retina is a challenge in the development of therapeutic drugs and imaging agents. This challenge arises because internalization of compounds into the brain and retina is restricted by the blood-brain barrier (BBB) and blood-retinal barrier (BRB), respectively. Simple and reliable in vivo assays are necessary to identify compounds that can easily cross the BBB and BRB. METHODS: We developed six fluorescent indoline derivatives (IDs) and examined their ability to cross the BBB and BRB in zebrafish by in vivo fluorescence imaging. These fluorescent IDs were administered to live zebrafish by immersing the zebrafish larvae at 7-8 days post fertilization in medium containing the ID, or by intracardiac injection. We also examined the effect of multidrug resistance proteins (MRPs) on the permeability of the BBB and BRB to the ID using MK571, a selective inhibitor of MRPs. RESULTS: The permeability of these barriers to fluorescent IDs administered by simple immersion was comparable to when administered by intracardiac injection. Thus, this finding supports the validity of drug administration by simple immersion for the assessment of BBB and BRB permeability to fluorescent IDs. Using this zebrafish model, we demonstrated that the length of the methylene chain in these fluorescent IDs significantly affected their ability to cross the BBB and BRB via MRPs. CONCLUSIONS: We demonstrated that in vivo assessment of the permeability of the BBB and BRB to fluorescent IDs could be simply and reliably performed using zebrafish. The structure of fluorescent IDs can be flexibly modified and, thus, the permeability of the BBB and BRB to a large number of IDs can be assessed using this zebrafish-based assay. The large amount of data acquired might be useful for in silico analysis to elucidate the precise mechanisms underlying the interactions between chemical structure and the efflux transporters at the BBB and BRB. In turn, understanding these mechanisms may lead to the efficient design of compounds targeting the brain and retina.

In vivo imaging of zebrafish retinal cells using fluorescent coumarin derivatives.

BACKGROUND: The zebrafish visual system is a good research model because the zebrafish retina is very similar to that of humans in terms of the morphologies and functions. Studies of the retina have been facilitated by improvements in imaging techniques. In vitro techniques such as immunohistochemistry and in vivo imaging using transgenic zebrafish have been proven useful for visualizing specific subtypes of retinal cells. In contrast, in vivo imaging using organic fluorescent molecules such as fluorescent sphingolipids allows non-invasive staining and visualization of retinal cells en masse. However, these fluorescent molecules also localize to the interstitial fluid and stain whole larvae. RESULTS: We screened fluorescent coumarin derivatives that might preferentially stain neuronal cells including retinal cells. We identified four coumarin derivatives that could be used for in vivo imaging of zebrafish retinal cells. The retinas of living zebrafish could be stained by simply immersing larvae in water containing 1µg/ml of a coumarin derivative for 30 min. By using confocal laser scanning microscopy, the lamination of the zebrafish retina was clearly visualized. Using these coumarin derivatives, we were able to assess the development of the zebrafish retina and the morphological abnormalities induced by genetic or chemical interventions. The coumarin derivatives were also suitable for counter-staining of transgenic zebrafish expressing fluorescent proteins in specific subtypes of retinal cells. CONCLUSIONS: The coumarin derivatives identified in this study can stain zebrafish retinal cells in a relatively short time and at low concentrations, making them suitable for in vivo imaging of the zebrafish retina. Therefore, they will be useful tools in genetic and chemical screenings using zebrafish to identify genes and chemicals that may have crucial functions in the retina.