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首页> 外文期刊>Mutation Research, A. Reviews in Genetic Toxicology >Optimization of the Salmonella/mammalian microsome assay for urine mutagenesis by experimental designs
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Optimization of the Salmonella/mammalian microsome assay for urine mutagenesis by experimental designs

机译：沙门氏菌/哺乳动物微粒体测定尿液致突变性的实验设计优化

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Assessing urine mutagenieity with the almonella mtitagenicity test, la often limited by the volumes of the samples. Optimization of the assay was performed with factorial and Doehlert designs. Two fractional factorial designs 2~(3-1) (3 factors, 4 experiments) were used to estimate the main effects of the percent S9 in the mix, the time of liquid incubation, the inoculum size and the growth conditions. A Doehlert design (3 factors, 13 experiments) was used to study the main effects and the interactions of the NADP, G6P and S9 in the mix the positive markers were benzo[a]pyrene (BaP, 0.3 mu g/plate) and a pool of smokers' urine (SU, 1.25 ml equivalent/plate), The response was limited to the induction factor (IF, number of induced revertantsumber ofspontaneous revertants) with Salmonella typhimurhim TA98. The optimal conditions for BaP were: a 60 min period of liquid incubation arid a volume of 0.1 ml (apprbx, 10~8 cells/plate) of an overnight culture grown in 50 ml of Nutrient Broth No, 2 from a250 ml flask. The S9 mix (0.1 ml, final volume) included 1.5% of S9, 1.0 mM NADP and 4.4 mM G6P. The maximal IF was 15.79. The optimal conditions for SU were a 60 min period of liquid incubation and a volume of 0.1 ml (approx, 10~8 cells/plate) of an overnight culture grown in 7 ml of Nutrient Broth No. 2 from a 20X 180 mm tube. The S9 mix (0.1 ml, final volume) Included: 4% S9, 4.2 mM NADP and 5.2 rHMl G6P. The maximal IF was 10.95. These optimal conditions did not modify the spontaneous frequencies ofthe tester strains: TA97a, TA98, TA100 and TA102, The dose-response curves of mutagenlc urine samples were found to be non-linear. This mlcromethod required 8-fold less urine sample and 12,5-fold less liver homogenate as compared to the standard plate incorporation assay and was from 6.2- to 11.8-fold more sensitive to evaluate urine mutagenicity. The sensitivity of this technique was found to be limited to individuals smoking more than approx. 5 cigarettes/day by the standard extraction-concentrationprocedure.
机译：用almonella致突变性测试评估尿液的诱变性，通常受样品量的限制。使用因子设计和Doehlert设计进行测定的优化。使用两个分数阶乘设计2〜（3-1）（3个因子，4个实验）来评估混合物中S9的百分比，液体孵育时间，接种量和生长条件的主要影响。采用Doehlert设计（3个因素，13个实验）研究了NADP，G6P和S9在混合物中的主要作用以及相互作用，阳性标记为苯并[a] py（BaP，0.3μg /板）和吸烟者尿液（SU，1.25 ml当量/板），该反应仅限于鼠伤寒沙门氏菌TA98的诱导因子（IF，诱导的逆转体数量/自发逆转体数量）。 BaP的最佳条件是：60分钟的液体温育和体积为0.1 ml的过夜培养物（在250 ml烧瓶中的50 ml Nutrient Broth No. 2中生长）过夜培养。 S9混合物（0.1 ml，最终体积）包括1.5％的S9、1.0 mM NADP和4.4 mM G6P。最大IF为15.79。 SU的最佳条件是60分钟的液体温育和体积为0.1 ml（约10-8个细胞/板）的过夜培养物，该培养物是从20X 180毫米试管中在7 ml 2号营养肉汤中生长的。 S9混合物（0.1 ml，最终体积）包括：4％S9、4.2 mM NADP和5.2 rHM1 G6P。最大IF为10.95。这些最佳条件并未改变测试菌株TA97a，TA98，TA100和TA102的自发频率。诱变尿液样品的剂量反应曲线被发现是非线性的。与标准板掺入法相比，这种方法需要的尿液样本少8倍，肝脏匀浆少12,5-倍，并且对尿液的致突变性敏感性要高6.2至11.8倍。发现这项技术的敏感性仅限于吸烟量超过约50％的个人。按标准提取浓度程序每天5支香烟。

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