Bignami M, Ficorella C, Dogliotti E, Norman R L, Kaighn M E, Saffiotti U
Cancer Res. 1984 Jun;44(6):2452-7.
Cytotoxicity, alkali-labile DNA lesions, ouabain resistance mutations, and neoplastic transformation were analyzed concurrently in the BALB/3T3 ClA31 -1-1 cell line treated with the alkylating agent N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) for different exposure times (15, 30, 60, 90, 120, and 240 min; 24, 48, and 72 hr). The half-life of MNNG in complete medium was approximately 70 min, both without cells and with cell numbers as used in the assays for cytotoxicity (2 X 10(2) cells/60-mm dish), transformation (1 X 10(4) cells/dish), and mutation (1 X 10(5) cells/dish). The cytotoxic effect of MNNG (0.5 or 2 micrograms/ml) appeared to be completed after an exposure time between 100 and 200 min. Maximal frequency of ouabain resistance mutations, however, was reached after a much shorter treatment time (30 to 60 min). Detection of DNA damage by alkaline elution analysis showed maximal increase in single-strand breaks already after treatment for 30 min. Exposures for 30 min followed by posttreatment incubation for 30 or 90 min showed active repair of single-strand breaks during these periods, indicative of an even balance between the additional MNNG-induced damage and its repair. Morphological transformation assays, at the same treatment times and concentrations used in the mutation assays, yielded frequency curves that reached their maxima 1 to 3 hr later than did the mutation frequencies. The ratio of transformation to ouabain resistance mutation frequencies was 3.7 for short treatment times (30 to 60 min), while it increased to more than 20 for exposure times of 240 min or longer. The temporal dissociation in the exposure times for maximal induction of mutation and transformation, observed with MNNG in this cell line, supports the hypothesis that a single gene mutational event is not sufficient to account for the full expression of neoplastic transformation.
在用烷化剂N-甲基-N'-硝基-N-亚硝基胍（MNNG）处理不同暴露时间（15、30、60、90、120和240分钟；24、48和72小时）的BALB/3T3 ClA31 -1-1细胞系中，同时分析了细胞毒性、碱不稳定DNA损伤、哇巴因抗性突变和肿瘤转化。在完全培养基中，无论有无细胞，以及在细胞毒性检测（2×10²个细胞/60毫米培养皿）、转化检测（1×10⁴个细胞/培养皿）和突变检测（1×10⁵个细胞/培养皿）中所使用的细胞数量情况下，MNNG的半衰期约为70分钟。MNNG（0.5或2微克/毫升）的细胞毒性作用在暴露100至200分钟后似乎完成。然而，哇巴因抗性突变的最大频率在更短的处理时间（30至60分钟）后达到。通过碱性洗脱分析检测DNA损伤表明，处理30分钟后单链断裂就已出现最大程度增加。暴露30分钟后再进行30或90分钟的处理后孵育，结果显示在此期间单链断裂有活跃修复，这表明MNNG诱导的额外损伤与其修复之间达到了平衡。在与突变检测相同的处理时间和浓度下进行的形态学转化检测，得到的频率曲线比突变频率晚1至3小时达到最大值。短处理时间（30至60分钟）时，转化与哇巴因抗性突变频率之比为3.7，而暴露240分钟或更长时间时，该比值增加到20以上。在此细胞系中用MNNG观察到的突变和转化最大诱导暴露时间的时间解离，支持了单基因突变事件不足以解释肿瘤转化完全表达的假说。
