| Budget Amount *help |
¥3,000,000 (Direct Cost: ¥3,000,000)
Fiscal Year 2004: ¥800,000 (Direct Cost: ¥800,000)
Fiscal Year 2003: ¥2,200,000 (Direct Cost: ¥2,200,000)
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| Research Abstract |
A variety of quantitative chemical assays have been used for the pharmacological study of drugs mainly using urine, blood and tissue homogenates from animals. Also, Autoradiography methods, which are commonly employed for pharmacological studies on drugs, are sensitive and specific, but possess drawbacks inherent in the use of a radioisotope. They also require time-consuming and complicated techniques, and only provide an indirect detection system for drug localization in cells and tissues. Autofluorescence is also used as a marker in the case of some drugs, but it is lacking in sensitivity. In order to overcome each of these drawbacks and develop an easy and generally applicable method revealing a precise localization or uptake of the drug in the cells and tissues, we established the present immunocytochemistry (ICC) method. In this study we used an anthracycline antibiotic daunomycin (DM) as a prototype. Successful DM ICC required a series of processes prior to the immunocytochemical
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reaction : the cells were first fixed with GA, then reducted with NaBH4, treated with hydrochloric acid, and finally digested with protease. The cell specimens were then subjected to immunoreaction with anti-DM serum followed by peroxidase-labeled goat anti-rabbit IgG/Fab', and in both the immune reagents the detergent Triton X-100 was contained as well. The present ICC covering all these processes successfully stained for DM in the nucleus and in the perinuclear Golgi region of the cytoplasm of the BD cells, this being consistent with the results obtained by the DM autofluorescence method. This ICC was found to be three times as sensitive as the cytofluorometric method, and applicable to the paraffin sections of the liver of rats 24 hr after an i.v. injection of DM. The principle used in the present study for developing DMD ICC might be applied to other drugs containing the primary amino group(s) in the molecule. Thus, these ICCs for drugs are direct, precise and easy new methods which should have potential for pharmacology and toxicolog studies of drugs, revealing the localization of a drug in cells and tissues. Less
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