Delivery Method, Target Gene Structure, and Growth Properties of Target Cells Impact Mutagenic Responses to Reactive Nitrogen and Oxygen Species

详细信息    查看全文

• 作者：Min Young Kim ; Chang Hoon Lim ; Laura J. Trudel ; William M. Deen ; Gerald N. Wogan
• 刊名：Chemical Research in Toxicology
• 出版年：2012
• 出版时间：April 16, 2012
• 年：2012
• 卷：25
• 期：4
• 页码：873-883
• 全文大小：366K
• 年卷期：v.25,no.4(April 16, 2012)
• ISSN：1520-5010

文摘

Dysregulated production of nitric oxide (NO^{鈥?/sup>) and reactive oxygen species (ROS) by inflammatory cells in vivo may contribute to mutagenesis and carcinogenesis. Here, we compare cytotoxicity and mutagenicity induced by NO鈥?/sup> and ROS in TK6 and AS52 cells, delivered by two methods: a well-characterized delivery system and a novel adaptation of a system for coculture. When exposed to preformed NO鈥?/sup>, a cumulative dose of 620 渭M min reduced the viability of TK6 cells at 24 h to 36% and increased mutation frequencies in the HPRT and TK1 genes to 7.7 脳 10鈥? (p < 0.05) and 24.8 脳 10鈥? (p < 0.01), 2.7- and 3.7-fold higher than background, respectively. In AS52 cells, cumulative doses of 1700 and 3700 渭M min reduced viability to 49 and 22%, respectively, and increased the mutation frequency 10.2- and 14.6-fold higher than the argon control (132 脳 10鈥? and 190 脳 10鈥?, respectively). These data show that TK6 cells were more sensitive than AS52 cells to killing by NO鈥?/sup>. However, the two cell lines were very similar in relative susceptibility to mutagenesis; on the basis of fold increases in MF, average relative sensitivity values [(MF_exp/MF_control)/cumulative NO鈥?/sup> dose] were 5.16 脳 10鈥? and 4.97 脳 10鈥? 渭M鈥? min鈥? for TK6 cells and AS52 cells, respectively. When AS52 cells were exposed to reactive species generated by activated macrophages in the coculture system, cell killing was greatly reduced by the addition of NMA to the culture medium and was completely abrogated by combined additions of NMA and the superoxide scavenger Tiron, indicating the relative importance of NO鈥?/sup> to loss of viability. Exposure in the coculture system for 48 h increased mutation frequency in the gpt gene by more than 9-fold, and NMA plus Tiron again completely prevented the response. Molecular analysis of gpt mutants induced by preformed NO鈥?/sup> or by activated macrophages revealed that both doubled the frequency of gene inactivation (40% in induced vs 20% in spontaneous mutants). Sequencing showed that base-substitution mutations dominated the spectra, with transversions (30鈥?0%) outnumbering transitions (10鈥?0%). Virtually all mutations took place at guanine sites in the gene. G:C to T:A transversions accounted for about 30% of both spontaneous and induced mutations; G:C to A:T transitions amounted to 10鈥?0% of mutants; insertions, small deletions, and multiple mutations were present at frequencies of 0鈥?0%. Taken together, these results indicate that cell type and proximity to generator cells are critical determinants of cytotoxic and genotoxic responses induced by NO鈥?/sup> and reactive species produced by activated macrophages.}