Intracellular localization of tetrodotoxins in pufferfish liver

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Intracellular localization of terodotoxins (TTXs) in pufferfish liver was investigated using three species of puffers (Takifugu pardalis, T.porphyreus, and T.vermicularis snyderi) with a variety of toxicity. Livers were minced and homogenized in a tissue homogenizer with 3 volumes of 20mM MOPS-KOH buffer (pH7.2) containing 60mM KCl, 180mM sucrose, 1mM EGTA,and 10muM phenylmethanesulfony fluoride (PMSF) at 200 rpm for 30 min. After removal of tissue debris, the homogenate was separated into five fractions (blood cell fr., nuclear fr., mitochondrial fr., microsomal fr., and cytosol fr.) by a centrifugation method.Toxins were extracted from each fraction by heating with 0.1% acetic acid in boiling water for 10 min. All the extracts from the five fractions showed lethal toxicity on mouse assay, although the toxin amount markedly varied from fraction to fraction. About 80% of the total toxin amounts was recovered in the cytosol fr., 1-10% each in the blood cell fr., the nuclear fr., and the mitochondrial fr., and about 1% in the microsomal fr., regardless of species and toxicity levels of livers. These results indicate that the toxins are widely distributed in organcella in liver cells.Analyzes by high performance liquid chromatogarphy (HPLC), gaschromatography-mass spectrometry, and liquid chromatography-fast atom bombardment mass spectrometry demonstrated that TTX was the major toxin principle in each fraction. In addition, the cytosol fr., even without the treatment with acetic acid, showed toxicity on mouse bioassay and the toxin in it was also found to be TTX by HPLC.It was concluded that TTX in the cytosol fr.exists in a free form, without binding to other substances.