Goijman S G, Frasch A C, Stoppani A O
Centro de Investigaciones Bioenergéticas, Facultad de Medicina, Universidad de Buenos Aires, Argentina.
Rev Argent Microbiol. 1984;16(2):75-86.
Incubation of Trypanosoma cruzi culture (epimastigote) forms with nifurtimox (10 or 100 microM), benznidazole (38 or 380 microM) and beta-lapachone (1.6 or 7.8 microM) produced damage of nuclear DNA, as shown by the increased rate of the "unscheduled DNA synthesis" in epimastigotes arrested at phase S (9-, 3-, and 6-fold, respectively). alpha-lapachone, a position isomer of beta-lapachone, was completely ineffective. In order to demonstrate the "unscheduled repair of DNA", the semiconservative replication was inhibited by preincubating the epimastigotes for 16 hours with 10 mM hydroxyurea and 0.3 mM cycloheximide. Kinetoplast DNA (kDNA) extracted from epimastigotes pretreated with the trypanocidal agents revealed an increased number of single-strand breaks. After alkaline agarose-gel electrophoresis, a fast moving DNA fraction was detected in the kDNA from nifurtimox, benznidazole and beta-lapachone-treated parasites, while trapping of alkali-denatured kDNA by nitrocellulose filters, was significantly increased after treating the epimastigotes with the same drugs. Reincubation of these epimastigotes in fresh medium for 24 h, reestablished kDNA electrophoretic and filtration patterns to normality, except with 7.8 microM beta-lapachone, thus proving the reversibility of DNA lesions. Redox-cycling of nifurtimox and beta-lapachone in T. cruzi generates oxygen radicals, and accordingly, the higher effectiveness of these drugs (as compared with benznidazole and alpha-lapachone) supports the role of oxygen radicals for the trypanocidal action.
用硝呋替莫（10或100微摩尔）、苯硝唑（38或380微摩尔）和β-拉帕醌（1.6或7.8微摩尔）培养克氏锥虫培养物（上鞭毛体），会导致核DNA损伤，这表现为处于S期的上鞭毛体中“非预定DNA合成”速率增加（分别增加9倍、3倍和6倍）。α-拉帕醌是β-拉帕醌的位置异构体，完全无效。为了证明“DNA的非预定修复”，通过将上鞭毛体与10毫摩尔羟基脲和0.3毫摩尔环己酰亚胺预孵育16小时来抑制半保留复制。从用杀锥虫剂预处理的上鞭毛体中提取的动基体DNA（kDNA）显示单链断裂数量增加。碱性琼脂糖凝胶电泳后，在经硝呋替莫、苯硝唑和β-拉帕醌处理的寄生虫的kDNA中检测到一个快速移动的DNA片段，而在用相同药物处理上鞭毛体后，硝酸纤维素滤膜对碱变性kDNA的捕获显著增加。将这些上鞭毛体在新鲜培养基中再孵育24小时后，除了用7.8微摩尔β-拉帕醌处理的情况外，kDNA的电泳和过滤模式恢复正常，从而证明了DNA损伤的可逆性。硝呋替莫和β-拉帕醌在克氏锥虫中的氧化还原循环会产生氧自由基，因此，这些药物（与苯硝唑和α-拉帕醌相比）更高的有效性支持了氧自由基在杀锥虫作用中的作用。
