目的 建立增强版细菌回复突变实验，并应用于N-亚硝基普萘洛尔（NNP）的致突变风险评价。方法 使用N-亚硝基二乙胺（NDEA）、N-亚硝基二甲胺（NDMA）、1-环戊基-4-亚硝基哌嗪（CPNP）比较不同预培养时间及不同S9含量对细菌回复突变实验结果的影响：采用鼠伤寒沙门氏菌（TA98、TA100、TA1535、TA1537）及大肠杆菌WP2 uvrA（pkM101）回复突变实验预培养法，NDEA和NDMA设置6个质量浓度（每皿5 000、2 500、1 000、500、250、125μg），并分别在10%大鼠S9/10%仓鼠S9/30%大鼠S9/30%仓鼠S9/非代谢活化条件下开展Ames实验；CPNP设置2个质量浓度（每皿500、125μg），在30%大鼠S9/30%仓鼠S9/非代谢活化条件下开展Ames实验。NDEA和NDMA与S9 mix或0.1 mol·L^-1缓冲液、细菌混合后置于恒温水浴摇床在37℃、50 r·min^-1预培养30或60 min（仅10%大鼠S9代谢活化条件下预培养60 min）,CPNP在相同设备条件下预培养30 min。NNP的致突变风险评价：NNP设置5个质量浓度（每皿62.500 0、31.250 0、15.625 0、7.812 5、3.900 0μg），并于30%大鼠S9、30%仓鼠S9及非代谢活化条件下进行，NNP与S9 mix或0.1 mol·L^-1缓冲液、菌株混合后置于恒温水浴摇床在37℃、50 r·min^-1预培养30 min。结果 所有代谢活化条件下，与溶媒对照组比较，NDEA可诱导TA98、TA100、TA1535和WP2 uvrA（pkM101）出现与剂量相关的回复突变菌落数增加；在仓鼠S9代谢活化条件下，NDMA可诱导TA100、TA1535和WP2 uvrA（pkM101）出现与剂量相关的回复突变菌落数增加。CPNP在非S9条件下可诱导TA1535和TA1537的各菌回复突变率明显增加；30%大鼠和仓鼠S9代谢条件下诱导TA100、TA1535、TA1537和WP2uvrA（pkM101）的菌落回复突变数明显增加。延长预培养时间（30～60 min）未增强NDEA/NDMA的代谢活化效果。增加大鼠S9含量（10%至30%）提高了NDEA对TA1535和TA1537的诱变性，也提高了NDMA对TA1537的诱变性，但降低了其对TA98、TA100和WP2 uvrA（pkM101）的诱变性；增加仓鼠S9含量仅提高NDEA对WP2 uvrA（pkM101）的诱变性。在仓鼠S9代谢活化条件下，NNP导致TA1535回复突变菌落数增加。结论 相同剂量条件下，CPNP诱导的回复突变菌落数量远超过NDEA和NDMA。预培养时间延长至60 min对代谢活化效果无进一步改进，S9的代谢活化条件与其含量成正相关，仓鼠S9的代谢活化效果优于大鼠S9。NNP的增强细菌回复突变实验结果为阳性，提示其代谢活化产物具有致突变性风险。

Objective To establish an enhanced bacterial reverse mutation test and apply it to the mutagenic risk assessment of Nnitroso-propranolol. Methods The effects of different pre-culture times and different S9 contents on the results of the bacterial reverse mutation test were compared using N-nitrosodiethylamine（NDEA）, N-nitrosodimethylamine（NDMA）, and 1-cyclopentyl-4-nitrosopiperazine（CPNP）. The reverse mutation experiments of Salmonella typhimurium（TA98, TA100, TA1535, TA1537） and Escherichia coli WP2 uvrA（pkM101） were conducted using the pre-culture method. For NDEA and NDMA, six mass concentrations（5 000, 2 500, 1 000, 500, 250, and 125 μg per plate） were set, and the Ames test was carried out under 10% rat S9/10% hamster S9/30% rat S9/30% hamster S9/non-metabolic activation conditions. For CPNP, two mass concentrations（500 and 125 μg per plate） were set, and the Ames test was carried out under 30% rat S9/30% hamster S9/non-metabolic activation conditions. NDEA and NDMA were mixed with S9 mix or 0.1 mol·L^-1 buffer solution and bacteria, and then pre-cultured for 30 or 60 min at 37 ℃ and 50 r·min^-1 in a constant temperature water bath shaker（only 60 min for 10% rat S9 metabolic activation condition）, while CPNP was pre-cultured for 30 min under the same conditions. The mutagenic risk assessment of NNP: NNP was set at five mass concentrations（62.500 0, 31.250 0, 15.625 0, 7.812 5, and 3.900 0 μg per plate）, and the Ames test was conducted under 30% rat S9, 30% hamster S9, and nonmetabolic activation conditions. NNP was mixed with S9 mix or 0.1 mol·L^-1 buffer solution and the strain, and then pre-cultured for 30 min at 37 ℃ and 50 r·min^-1 in a constant temperature water bath shaker. Results Under all metabolic activation conditions, NDEA could induce a dose-related increase in the number of revertant colonies in all strains except TA98, TA100, TA1535 and WP2 uvrA（pkM101）. Under hamster S9 metabolic activation conditions, NDMA could induce a dose-related increase in the number of revertant colonies in all strains except TA100, TA1535 and WP2 uvrA（pkM101）. CPNP could induce a significant increase in the reverse mutation rate of TA1535 and TA1537 under non-S9 conditions; at 30% rat and hamster S9 metabolic conditions, it could induce a significant increase in the number of revertant colonies in TA100, TA1535, TA1537 and WP2 uvrA（pkM101）. Extending the preincubation time from 30 to 60 minutes did not enhance the metabolic activation effect of NDEA/NDMA. Increasing the S9 content in rats（from 10% to 30%） enhanced the mutagenicity of NDEA towards TA1535 and TA1537, and also increased the mutagenicity of NDMA towards TA1537, but reduced its mutagenicity towards TA98, TA100, and WP2 uvrA（pkM101）. but reduced it on TA98, TA100 and WP2 uvrA; increasing the content of hamster S9 only enhanced the mutagenicity of NDEA on WP2 uvrA. Under hamster S9 metabolic activation conditions, 3.9 to 62.5 μg per plate of N-nitroso-propranolol could cause an increase in the number of revertant colonies in TA1535. Conclusion Under the same dose conditions, the number of revertant colonies induced by CPNP far exceeds that of NDEA and NDMA. Extending the pre-incubation time to 60 minutes did not further improve the metabolic activation effect, and the metabolic activation effect of S9 was positively correlated with its content. The metabolic activation effect of hamster S9 was better than that of rat S9. The result of the enhanced bacterial reverse mutation test for NNP is positive, indicating that its metabolically activated products have a mutagenic risk.

R965

药品监管科学全国重点实验室课题“药品杂质遗传毒性评价新技术和生物标志物研究”(2023SKLDRS0128)