Dackiewicz P, Skladanowski A, Konopa J
Department of Pharmaceutical Technology and Biochemistry, Technical University of Gdansk, Poland.
Chem Biol Interact. 1995 Nov 17;98(2):153-66. doi: 10.1016/0009-2797(95)03643-1.
DNA adduct formation by enzymatically activated mitoxantrone and ametantrone has been studied by 32P-postlabelling method. Both drugs were activated by peroxidase/hydrogen peroxide system and formed several DNA adducts when reacted with calf thymus DNA. Mitoxantrone gave 3 and ametantrone 4 different DNA adducts with apparently similar chromatographic features suggesting that DNA adducts formed by both compounds do not differ significantly in their chemical structure. Despite this similarity, the level of DNA modification is 10 times higher for mitoxantrone compared to ametantrone. We did not observe DNA adducts in control samples where both drugs were incubated with DNA in the absence of the activating system. It indicates the importance of oxidative activation of mitoxantrone and ametantrone for their ability to bind covalently DNA. In order to identify nucleobases involved in the formation of DNA adducts by anthracenediones, polydeoxyadenosine, polydeoxythymidine, deoxyguanosine 3'-monophosphate and deoxycytosine 3'-monophosphate were modified by mitoxantrone and ametantrone activated in the above mentioned oxidating system. We proved that the only nucleobase modified by both drugs is guanine with no alkylation observed at other DNA bases. The pattern of adducts formed with deoxyguanosine 3'-monophosphate is reminiscent of that obtained with calf thymus DNA. In addition, mitoxantrone was found to be phosphorylated during the postlabelling procedure, most probably at the 1,4-hydroxyl groups of the chromophore. Ametantrone which does not possess hydroxyl groups attached to the chromophore core was resistant to phosphorylation by T4 polynucleotide kinase and gamma-[32P]ATP. These results for the first time provide direct evidence that mitoxantrone and ametantrone form DNA adducts when activated by oxidation in vitro.
通过³²P后标记法研究了酶促活化的米托蒽醌和氨茴环素形成DNA加合物的情况。两种药物均由过氧化物酶/过氧化氢系统活化，与小牛胸腺DNA反应时形成了几种DNA加合物。米托蒽醌产生了3种，氨茴环素产生了4种不同的DNA加合物，其色谱特征明显相似，这表明两种化合物形成的DNA加合物在化学结构上没有显著差异。尽管有这种相似性，但米托蒽醌导致的DNA修饰水平比氨茴环素高10倍。在对照样品中，当两种药物在没有活化系统的情况下与DNA一起孵育时，我们未观察到DNA加合物。这表明米托蒽醌和氨茴环素的氧化活化对于它们与DNA共价结合的能力很重要。为了鉴定蒽二酮形成DNA加合物所涉及的核碱基，用上述氧化系统中活化的米托蒽醌和氨茴环素对聚脱氧腺苷、聚脱氧胸苷、脱氧鸟苷3'-单磷酸和脱氧胞苷3'-单磷酸进行了修饰。我们证明，两种药物唯一修饰的核碱基是鸟嘌呤，在其他DNA碱基上未观察到烷基化。用脱氧鸟苷3'-单磷酸形成的加合物模式让人想起用小牛胸腺DNA获得的模式。此外，发现米托蒽醌在后标记过程中被磷酸化，最有可能是在发色团的1,4-羟基处。氨茴环素的发色团核心没有连接羟基，对T4多核苷酸激酶和γ-[³²P]ATP的磷酸化具有抗性。这些结果首次提供了直接证据，表明米托蒽醌和氨茴环素在体外被氧化活化时会形成DNA加合物。
