Mitomycin C-induced Cytogenotoxicity in Cultured Cells and Cardioprotective Effect of Luteolin During Ischemia- Reperfusion Injury in Rats

• Main Authors: Pei-Hu, 廖培湖
• Other Authors: 林瑞生
• Format: Others
• Language: zh-TW
• Published: 2012
• Online Access: http://ndltd.ncl.edu.tw/handle/49923085133743482702

博士 === 中山醫學大學 === 醫學研究所 === 100 === CHO cell, V79 cell and human T-lymphocytes are often used for investigating the cytogenotoxicity of cells in chemical substance in vitro studies. The cytogenotoxicity test methods applied in this study include micronuclei (MN), sister chromatid exchanges (SCEs) and chromosome aberrations (CAs). Mitomycin C (MMC) is an alkylating agent that causes DNA to cross-link and it is used for fighting cancer clinically but may lead to cancer in the long term or in excessive amounts. This study discusses the sensitivity difference of the three cultured cells with MMC-induced cytogenotoxicity and the addition of cysteine aspartic acid-specific protease (caspase-3). Among the three cultured cells, T-lymphocytes presented the highest sensitivity to the MMC-induced MN, CAs and SCEs cytogenotoxicity. CHO cell and V79 cell were different toward the sensitivity of the three test methods: CHO cell was more sensitive than V79 cell in MN and CAs tests, and had sensitivity equal to V79 cell in SCEs test. The three cultured cells also showed differences after CAs was further analyzed: in chromatid breaks and dicentrics, T-lymphocytes were the most sensitive and CHO cell was more sensitive than V79 cell; in acentrics and chromosome interchanges, the sensitivity of T-lymphocytes were much higher than the other two cells. We further discovered that the increment of caspase-3 activity played an important role in MMC-induced cytogenotoxicity. The MMC-induced increment of caspase-3 activity led to higher sensitivity in T-lymphocytes than CHO cell and V79 cell. Luteolin is a flavonoid in vegetables and is one of the natural polyphenol compounds. For example, Luteolin exists in fruit shell of Arachis hypogaea, Ajuga decumbus, Lonicera japonica Thunb, Gentianopsis paludosa, and Valeriana amurensis Smir. Previous studies indicated that antioxidants can reduce effectively myocardial ischemia-reperfusion (IR) injury. Luteolin is an antioxidative flavonoid, thus this study mainly investigates whether luteolin has protective effects on IR injuries of rats’ hearts. Using rats as samples, this study observed the myocardial injuries of ischemia and coronary artery reperfusion caused by ligation of coronary artery to evaluate the heart protecting effect of Luteolin. Luteolin was injected into a rat''s veins 15min before the coronary artery ligation, and the results showed that, when ischemia occurred, the pretreatment by luteolin (10μg/kg) can significantly decrease the incidents and duration of ventricular tachycardia (VT) and ventricular fibrillation (VF) and the death rate of rats. The luteolin (1μg/kg) also lowered the death rate and ventricular arrhythmia in the coronary artery reperfusion phase. Using luteolin can also reduce the contents of lactate dehydrogenase and NO. In biological statistics, the Luteolin (10μg/kg) narrowed the range of myocardial infarction and reduced the malondialdehyde (MDA) yield of myocardial IR injury tissue samples. We further confirmed that luteolin can decease inducible NO synthase (iNOS) proteins and mRNA (imRNA) expression, but cannot change significantly the expression of neuronal NO synthase (nNOS) or endothelial NO synthase (eNOS). Conclusion:The MMC in the three cultured cells can at least partially increase frequencies of MN, CAs and SCEs by activating caspase-3. As T-lymphocytes presented the highest precision, positive prediction and sensitivity, they are the most suitable samples for studying genotixicity. Luteolin can protect cardiac muscles against IR injuries. At least part of the myocardial protecting effect of Luteolin is through restraining iNOS to reduce formation of NO and by its own antioxidative effect.