Cloutier J F, Castonguay A
Laboratory of Cancer Etiology and Chemoprevention, Faculty of Pharmacy, Laval University, Quebec City, Canada.
Chem Biol Interact. 1998 Mar 12;110(1-2):7-25. doi: 10.1016/s0009-2797(97)00114-2.
The tobacco specific nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) is present in tobacco smoke and is hepatocarcinogenic in rats. Its bioactivation in rat hepatocytes leads to methylation and pyridyloxobutylation of DNA. Rat hepatocytes were cultured in serum-free William medium E on collagen-coated dishes. We demonstrated that some enzymes of the base and/or excision-repair pathways were involved in repair of NNK-induced DNA damage, measured by [methyl-3H] thymidine incorporation. Unscheduled DNA synthesis (UDS) induced by N-methyl-N-nitrosourea (MNU), NNK, N'-nitrosonornicotine (NNN) and 4-(acetoxymethylnitrosamino)-1-(3-pyridyl)-1-butanone (NNKOAc) increased 2.9-, 2.8-, 1.5- and 3.5-fold, respectively, suggesting that methylated and/or pyridyloxobutylated-DNA by these four nitroso compounds is repaired by the excision pathway. Moreover, levels of NNK-induced UDS were dose (1-3 mM) and time (1-18 h) dependent. Enzymes involved in the excision repair pathways were selectively inhibited. Inhibitors of DNA topoisomerase I (camptothecin) and topoisomerase II (etoposide, nalidixic acid) did not decrease the induction of UDS, suggesting that topoisomerases are not involved in the repair of NNK-induced damage. While aphidicolin and arabinocytidine (DNA polymerase alpha, delta, epsilon inhibitors) totally inhibited NNK- and NNKOAc-induced UDS, dideoxythymidine (DNA polymerase beta inhibitor) inhibited NNK- and NNKOAc-induced UDS by 40 and 33%, respectively. We conclude that DNA polymerase alpha, delta or epsilon and to a lesser degree polymerase beta are involved in the repair of pyridyloxobutylated DNA. Previous studies showed that inhibition of poly(ADP-ribosyl) polymerase (PARP) by 3-aminobenzamide (3-ab) facilitated DNA ligation. Our results demonstrate that 3-ab increased NNK-induced UDS, but does not affect NNKOAc-induced UDS. These observations suggest that the ligation step is rate limiting in the repair of methylated DNA but not of pyridyloxobutylated DNA.
烟草特有的亚硝胺4-(甲基亚硝胺基)-1-(3-吡啶基)-1-丁酮(NNK)存在于烟草烟雾中，对大鼠具有肝致癌性。它在大鼠肝细胞中的生物活化导致DNA的甲基化和吡啶氧基丁基化。大鼠肝细胞在无血清的威廉培养基E中，于胶原包被的培养皿上培养。我们证明，通过[甲基-3H]胸苷掺入法测定，碱基和/或切除修复途径的一些酶参与了NNK诱导的DNA损伤的修复。由N-甲基-N-亚硝基脲(MNU)、NNK、N'-亚硝基降烟碱(NNN)和4-(乙酰氧基亚硝胺基)-1-(3-吡啶基)-1-丁酮(NNKOAc)诱导的非预定DNA合成(UDS)分别增加了2.9倍、2.8倍、1.5倍和3.5倍，这表明这四种亚硝基化合物导致的甲基化和/或吡啶氧基丁基化的DNA通过切除途径进行修复。此外，NNK诱导的UDS水平呈剂量(1-3 mM)和时间(1-18小时)依赖性。参与切除修复途径的酶被选择性抑制。DNA拓扑异构酶I(喜树碱)和拓扑异构酶II(依托泊苷、萘啶酸)的抑制剂并没有降低UDS诱导，这表明拓扑异构酶不参与NNK诱导损伤的修复。虽然阿非科林和阿糖胞苷(DNA聚合酶α、δ、ε抑制剂)完全抑制了NNK和NNKOAc诱导的UDS，但双脱氧胸苷(DNA聚合酶β抑制剂)分别抑制了NNK和NNKOAc诱导的UDS的40%和33%。我们得出结论，DNA聚合酶α、δ或ε以及程度较轻的聚合酶β参与了吡啶氧基丁基化DNA的修复。先前的研究表明，3-氨基苯甲酰胺(3-ab)对聚(ADP-核糖基)聚合酶(PARP)的抑制促进了DNA连接。我们的结果表明，3-ab增加了NNK诱导的UDS，但不影响NNKOAc诱导的UDS。这些观察结果表明，连接步骤在甲基化DNA的修复中是限速步骤，但在吡啶氧基丁基化DNA的修复中不是。
