[3H]-(N-正丙基)卟吩霉素（POR）被用来检测和识别在不同条件下活体EMT6小鼠乳腺肿瘤细胞中形成的单个和双个标记物加合物。为了提供真实的标准加合物，小牛胸腺DNA被用还原活化的POR处理，然后被消化成核苷和POR-核苷加合物。结果得到了三种主要加合物，并进行了鉴定。其中两种是单加合物，由去氧鸟苷连接在它的N2位于的C-1和10-脱十氨酰基 POR。第三种是双加合物，它将POR交联到两个去氧鸟苷的N2位于上。经过[3H]-POR处理的EMT6细胞中的DNA被消化并通过HPLC分析。DNA关联标签位于胸腺嘧啶和两种单加合物和一种双加合物，与上述一致。胸腺嘧啶中的标签来源于POR的N-去甲基化，并重新将标签加入新的嘧啶酸残基中。在缺氧条件下形成的加合物比在氧气中形成的更丰富。此外，单加合物与交联比例不同，分别为缺氧和有氧细胞约为1:1和2:1。空气和缺氧下烷基化的不同模式可能与POR在缺氧下更大的毒性有关。当细胞同时与POR和双香豆素处理时，加合物水平较低，并且在缺氧情况下主要观察到一种新的未知加合物；这些变化可能与双香豆素存在时POR的毒性改变有关。HPLC测定同时检测到DNA中全部稳定的单个和双个加合物，具有很好的灵敏度（大于或等于2 x 10（6）加合物/核苷酸）和优异的重现性。当具有高比活度的MC或POR可以被用来处理时，此方法应该可以普遍适用于所有细胞和组织。

[3H]-(N-la-methyl) Porfiromycin (POR) was employed to detect and identify the radiolabeled mono- and bis-adducts formed in living EMT6 mouse mammary tumor cells under different conditions. To provide authentic standard adducts, calf-thymus DNA was treated with POR under reductive activation, then digested to nucleosides and POR-nucleoside adducts. The three major adducts formed were isolated by HPLC and authenticated. Two were mono-adducts, composed of deoxyguanosine linked at its N2-position to C-1 of POR and of 10-decarbamoyl POR. The third was a bis-adduct, in which POR was crosslinked to two deoxyguanosines at their N2-positions. DNA from [3H]-POR treated EMT6 cells was digested an analyzed by HPLC. DNA-associated label was located in thymidine and in two mono-adducts and one bis-adduct identical to those described above. Label in thymidine resulted from N-demethylation of POR and reincorporation of label into new thymidylate residues. Adducts were formed more abundantly in hypoxia than in air. In addition, the mono-adduct to crosslink ratios were different, approximately 1:1 and 2:1 for hypoxic and aerobic cells, respectively. The different patterns of alkylation in air and hypoxia may be related to the greater toxicity of POR in hypoxia. When cells were treated simultaneously with POR and dicumarol, adduct levels were lower, and a new, unknown adduct was observed primarily under hypoxia; these changes may be related to the altered toxicity of POR in the presence of dicumarol. The HPLC assay detected simultaneously the full array of stable mono- and bis-adducts in DNA with good sensitivity (greater than or equal to 2 x 10(6) adducts/nucleotide) and excellent reproducibility. This assay should be generally applicable to all cells and tissues when MC or POR with high specific radioactivity can be employed.