Kim Min Young, Wogan Gerald N
Biological Engineering Division and Chemistry Department, Massachusetts Institute of Technology, 77 Massachusetts Avenue, Cambridge, Massachusetts 02139, USA.
Chem Res Toxicol. 2006 Nov;19(11):1483-91. doi: 10.1021/tx0601354.
Dysregulated production of nitric oxide (NO*) and reactive oxygen species by inflammatory cells contributes to mutagenesis and carcinogenesis. We have characterized mutagenesis in the target supF gene of pSP189 replicating in AD293 cells cocultivated with mouse macrophage-like RAW264.7 cells activated with interferon-gamma (IFN-gamma) and lipopolysaccharide (LPS). Activated macrophages produced substantial amounts of NO*, superoxide anion (O2*-), and hydrogen peroxide (H2O2) over 12-72 h periods. A time-dependent decrease in total cell number and a 3.7-fold increase in supF mutation frequency (MF), compared with unstimulated controls, were observed at 72 h. The increase in MF was effectively suppressed by N-methyl-L-arginine monoacetate (NMA), an NO* synthase inhibitor, and also by superoxide dismutase (SOD) and catalase (CAT); cotreatment with NMA and SOD/CAT suppressed mutagenesis by 87% at 72 h. Mutations in supF were mainly multiple sequence changes (47%) and single base pair substitutions (51%) following IFN-gammaLPS activation. Following cotreatment with NMA alone or together with SOD/CAT, however, single base pair substitutions were prevalent (70 and 85%); decreased multiple mutations were observed (24 and 11%). Almost all single base pair substitutions induced under all exposure conditions occurred at G:C base pairs (87.8-94.6%). Whereas those induced by all treatments consisted predominantly of G:C to T:A transversions, G:C to T:A and A:T to T:A transversions were less frequent following treatment with NMA alone or with SOD/CAT compared to those induced by activated macrophages without additional treatment. Our results strongly suggest that ONOO- or its derivatives generated by reaction of NO* with O2*- may have been a major contributor to the observed mutagenesis by the activated macrophages, and mitigating their effects might serve a preventive function in ameliorating cancer risks associated with prolonged inflammation.
炎症细胞中一氧化氮（NO*）和活性氧的产生失调会导致诱变和致癌作用。我们已经对在与经γ干扰素（IFN-γ）和脂多糖（LPS）激活的小鼠巨噬细胞样RAW264.7细胞共培养的AD293细胞中复制的pSP189靶标supF基因中的诱变进行了表征。活化的巨噬细胞在12至72小时内产生了大量的NO*、超氧阴离子（O2*-）和过氧化氢（H2O2）。与未刺激的对照相比，在72小时时观察到总细胞数随时间减少，supF突变频率（MF）增加了3.7倍。MF的增加被NO合酶抑制剂N-甲基-L-精氨酸单乙酸盐（NMA）以及超氧化物歧化酶（SOD）和过氧化氢酶（CAT）有效抑制；在72小时时，NMA与SOD/CAT联合处理可将诱变抑制87%。在IFN-γLPS激活后，supF中的突变主要是多序列变化（47%）和单碱基对替换（51%）。然而，在单独用NMA或与SOD/CAT联合处理后，单碱基对替换很普遍（分别为70%和85%）；观察到多突变减少（分别为24%和11%）。在所有暴露条件下诱导的几乎所有单碱基对替换都发生在G:C碱基对处（87.8 - 94.6%）。虽然所有处理诱导的那些主要由G:C到T:A的颠换组成，但与未进行额外处理的活化巨噬细胞诱导的相比，单独用NMA或与SOD/CAT处理后，G:C到T:A和A:T到T:A的颠换频率较低。我们的结果强烈表明，NO与O2*-反应产生的过氧亚硝酸盐（ONOO-）或其衍生物可能是活化巨噬细胞观察到的诱变的主要促成因素，减轻它们的影响可能在改善与长期炎症相关的癌症风险方面起到预防作用。
