Fusako Takayama*, Toru Egashira and Yasumitsu Yamanaka
Department of Pharmacology, Oita Medical University, 1-1, Idaigaoka, Hasama-machi, Oita 879-5593, Japan
*Corresponding author. FAX: +81-97-586-5729
Abstract: To establish a simple screening system for estimating efficacy of an agent for an oxidative-related lesion, we investigated the damage in isolated rat hepatocytes exposed to 75 mM tert-butyl hydroperoxide (t-BuOOH) and then subsequently incubated the cells in fresh medium. By electron spin resonance spectroscopy analysis using 5,5-dimethyl-1-pyrroline-N-oxide (DMPO), DMPO adducts of tert-butoxyl radicals and carbon center radicals were detected during the t-BuOOH exposure, and DMPO-OH formation was detected after t-BuOOH removal. In t-BuOOH-exposed cells, the level of phosphatidylcholine hydroperoxide (PCOOH), a peroxidative product of biomembranes in the hepatocytes, and the leakage of enzymes into the culture medium were significantly increased. An increase in acid phosphatase (AP) activity representing lysosome destabilization preceded the aspartate oxoglutarate aminotransferase (AST), alanine oxoglutarate aminotransferase (ALT) and lactate dehydrogenase (LDH) leakage. Ninjin-yoei-to added to the culture medium following the t-BuOOH exposure significantly inhibited the PCOOH formation and the leakage of AP, AST, ALT and LDH, concentration-dependently. Ninjin-yoei-to at 1 mg/ml in culture medium completely diminished these increases in enzyme activities down to the background levels found in control experiments and this reduction was greater than the most effective a-tocopherol concentration of 20 mmol/ml. Considering all of these results, it is likely Ninjin-yoei-to may exert its protective effect by anti-oxidative action and membrane stabilization.
Keywords: Ninjin-yoei-to, Biomembrane peroxidation, Oxidative stress, Lysosome, Isolated hepatocyte
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