Induction of Autophagy by Amino Acid Starvation in Fish Cells

Autophagy is well established as a starvation-induced process in yeast and mammalian cells and tissues. To elucidate the cellular mechanisms induced by starvation in fish, we characterized the induction of autophagy in cultured zebrafish cells under starvation conditions. As an autophagic marker protein, the microtubule-associated protein 1-light chain 3B protein (MAP1-LC3B) was cloned from the fish cells, and its expression and localization were characterized. In zebrafish embryonic (ZE) cells, posttranslational modifications produced two distinct forms of MAP1-LC3B, i.e., a cytosolic form and a membrane-bound form (types I and II, respectively). Immunofluorescence microscopy revealed fluorescently labeled autophagosomes in cells stably transfected with a green fluorescent protein (GFP)–MAP1-LC3B fusion protein and showed that this protein accumulated in punctate dots in a time-dependent manner in response to amino acid starvation. Starvation also induced the degradation of long-lived proteins. Treatment with 3-methyladenine and wortmannin, two class-III inhibitors of phosphoinositide 3-kinase (PI3K), repressed autophagy under starvation conditions, indicating that the PI3K class-III pathway regulates starvation-induced autophagy in fish

De Novo Accumulation of Tetrodotoxin and Its Analogs in Pufferfish and Newt and Dosage-Driven Accumulation of Toxins in Newt: Tissue Distribution and Anatomical Localization

The present study was undertaken to determine the amounts of tetrodotoxin (TTX) and its analogs (TTXs) in various tissues of toxin-bearing pufferfish (Canthigaster revulata and Takifugu flavipterus) and newt (Cynops pyrrhogaster) using specific polyclonal antibodies against TTXs, and to compare the obtained results with those mainly determined by high-performance liquid chromatography with fluorescence detection (HPLC-FLD). The anatomical localization of TTXs in these animals was also demonstrated immunohistochemically using the above-mentioned antibody. The ratio of the total amount of TTXs determined by ELISA to that determined by HPLC-FLD changed depending on the tissues examined in pufferfish. Such differences were also observed with the newt in tissue- and individual-dependent manners. Furthermore, TTXs, as well as decarbamoylsaxitoxin (dcSTX), an analog of saxitoxin (STX), were traced for their dynamic changes in tissue distribution, when the newt was fed authentic toxins or toxic animal tissues exogenously, demonstrating that a TTX analog, 5,6,11-trideoxyTTX, and dcSTX were not metabolized into TTX or STX. TTXs-immunoreactive (ir) staining was observed in the pancreas region of the hepatopancreas, the oocytes at the perinucleolus stage, the sac-like tissues just outside the serous membrane of the intestine, and the gland-like structure of the skin, but not in the muscles of pufferfish. TTXs-ir staining was also detected in the mature glands in the dermis of the adult and regenerated tail, but not in the liver, intestine, testis and ovary of the adult newt. TTXs-ir staining was detected in the epithelial cells of the intestine, the ovary, the mucous cells, and the dermis of the TTXs-administered newt. These results suggest that TTXs absorbed from the environment are distributed to various organs or tissues in a species-specific manner, regardless of whether or not these are metabolized in the bodies of toxin-bearing animals

Selenoneine, a Novel Selenium-Containing Compound, Mediates Detoxification Mechanisms against Methylmercury Accumulation and Toxicity in Zebrafish Embryo

The selenium (Se)-containing antioxidant selenoneine (2-selenyl-N(α),N(α),N(α)-trimethyl-l-histidine) has recently been discovered to be the predominant form of organic Se in tuna blood. Although dietary intake of fish Se has been suggested to reduce methylmercury (MeHg) toxicity, the molecular mechanism of MeHg detoxification by Se has not yet been determined. Here, we report evidence that selenoneine accelerates the excretion and demethylation of MeHg, mediated by a selenoneine-specific transporter, organic cations/carnitine transporter-1 (OCTN1). Selenoneine was incorporated into human embryonic kidney HEK293 cells transiently overexpressing OCTN1 and zebrafish blood cells by OCTN1. The K(m) for selenoneine uptake was 13.0 μM in OCTN1-overexpressing HEK293 cells and 9.5 μM in zebrafish blood cells, indicating high affinity of OCTN1 for selenoneine in human and zebrafish cells. When such OCTN1-expressing cells and embryos were exposed to MeHg–cysteine (MeHgCys), MeHg accumulation was decreased and the excretion and demethylation of MeHg were enhanced by selenoneine. In addition, exosomal secretion vesicles were detected in the culture water of embryos that had been microinjected with MeHgCys, suggesting that these may be responsible for MeHg excretion and demethylation. In contrast, OCTN1-deficient embryos accumulated MeHg, and MeHg excretion and demethylation were decreased. Furthermore, Hg accumulation was decreased in OCTN1-overexpressing HEK293 cells, but not in mock vector-transfected cells, indicating that selenoneine and OCTN1 can regulate MeHg detoxification in human cells. Thus, the selenoneine-mediated OCTN1 system regulates secretory lysosomal vesicle formation and MeHg demethylation. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1007/s10126-013-9508-1) contains supplementary material, which is available to authorized users