Gupta R C, Garg A, Earley K, Agarwal S C, Lambert G R, Nesnow S
Department of Preventive Medicine and Environmental Health, University of Kentucky, Lexington 40506.
Cancer Res. 1991 Oct 1;51(19):5198-204.
We have studied adduct formation of the antineoplastic agent diaziquone (AZQ; NSC 182986) with DNA and nucleotides in vitro. The aziridine moieties of AZQ can be expected to interact covalently with DNA which, in turn, presumably elicits the antitumor activity. We analyzed AZQ-DNA adducts by a modified 32P-postlabeling assay involving purification of the nuclease P1-enriched labeled adducts by high-salt C18 reversed-phase thin-layer chromatography and separation of the eluted adducts on a polyethyleneimine-cellulose layer using non-urea salt solutions. Modification of calf thymus DNA with AZQ produced two major (22% and 40%) and at least eight minor adducts. At equal concentrations of AZQ and DNA (1 micrograms/microliters each), peak binding was observed in about 2 h [1926 +/- 378 (SD) fmol/micrograms of DNA] with the binding levels remaining practically unchanged through 4 h. However, incubation for 24 h resulted in over 40% decline, indicating adduct instability. AZQ was found to be highly reactive in vitro as evidenced by its substantial binding (49 +/- 14 fmol/micrograms of DNA) even at a DNA:AZQ ratio of 100:1. When incubated with mononucleotides, AZQ reacted extensively with adenine, guanine, and cytosine but only slightly with thymine. Cochromatography of the modified DNA and nucleotides revealed that one of the major adducts and several minor adducts were guanine derived. The aziridine rings of AZQ were found to be the main reactive sites as its monoaminoalcohol derivative showed as much DNA reactivity as did the parent compound, but no activity was observed when both aziridine groups were hydrolyzed to diaminoalcohols. The improved 32P-postlabeling assay seems capable of detecting relatively polar adducts such as those formed with AZQ at a level of one adduct/10(9) nucleotides.
我们已经在体外研究了抗肿瘤药物重氮醌（AZQ；NSC 182986）与DNA和核苷酸的加合物形成情况。AZQ的氮丙啶部分有望与DNA发生共价相互作用，进而可能引发抗肿瘤活性。我们通过一种改良的³²P后标记分析法分析AZQ-DNA加合物，该方法包括通过高盐C18反相薄层色谱法纯化富含核酸酶P1的标记加合物，并使用非尿素盐溶液在聚乙烯亚胺-纤维素层上分离洗脱的加合物。用AZQ修饰小牛胸腺DNA产生了两种主要加合物（分别为22%和40%）以及至少八种次要加合物。在AZQ和DNA浓度相等（均为1微克/微升）时，约2小时观察到峰值结合（1926±378（标准差）飞摩尔/微克DNA），且在4小时内结合水平基本保持不变。然而，孵育24小时导致结合水平下降超过40%，表明加合物不稳定。即使在DNA与AZQ的比例为100:1时，AZQ在体外也具有高反应活性，这可通过其大量结合（49±14飞摩尔/微克DNA）得到证明。当与单核苷酸孵育时，AZQ与腺嘌呤、鸟嘌呤和胞嘧啶广泛反应，但与胸腺嘧啶反应轻微。修饰后的DNA和核苷酸的共色谱分析表明，主要加合物之一和几种次要加合物源自鸟嘌呤。发现AZQ的氮丙啶环是主要反应位点，因为其单氨基醇衍生物与母体化合物表现出相同的DNA反应活性，但当两个氮丙啶基团都水解为二氨基醇时未观察到活性。改进的³²P后标记分析法似乎能够检测相对极性的加合物，例如与AZQ形成的加合物，检测水平可达每10⁹个核苷酸一个加合物。
