The VITOTOX(R) test, an SOS bioluminescence Salmonella typhimurium test to measure genotoxicity kinetics
The VITOTOX(R) test, an SOS bioluminescence Salmonella typhimurium test to measure genotoxicity kinetics
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DOI：
10.1016/s1383-5718(96)00158-1
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发表时间：
1997-03-17
影响因子：
1.9
通讯作者：
Verschaeve, L
中科院分区：
文献类型：
--
作者：
vanderLelie, D;Regniers, L;Verschaeve, L
开发了一种检测遗传毒性的新测试，我们将其称为 VITOTOX(R) 测试。构建了基于大肠杆菌 recN 启动子的四种基因融合体，并评估了它们的 SOS 反应依赖性诱导。将野生型recN启动子、第二个LexA结合位点突变的衍生物、具有突变-35区域的衍生物、以及第二个LexA结合位点和-35区域都突变的衍生物克隆到pMOL877的无启动子费氏弧菌luxCDABE操纵子的上游，使得lux处于recN启动子衍生物的转录控制之下。在大肠杆菌和Ames测试鼠伤寒沙门氏菌菌株TA98、TA100和TA104中测试了启动子构建体的SOS应答的诱导能力。在所有菌株中，使用质粒 pMOL1067 和 pMOL1068 观察到最高的灵敏度和诱导，这两个质粒分别含有在第二个 LexA 结合位点突变的 recN 启动子或具有突变的 -35 区域的 recN 启动子控制下的 lux 操纵子。因此，含有pMOL1067或pMOL1068的菌株进一步用于遗传毒性测试。通过 VITOTOX 测试，可在 1-4 小时内检测到遗传毒性。 VITOTOX 测试非常敏感：对于大多数测试产品，最小可检测浓度 (MDC) 值比 Ames 测试和 SOS 比色测试中描述的值低得多（5 至 > 100 倍）。观察到与 Ames 试验结果有良好的相关性，但某些在 Ames 试验中不具有致突变性的 PAH 在 VITOTOX 试验中却具有遗传毒性。利用 VITOTOX 菌株，可以确定 SOS 诱导的动力学。这一特征使得区分基因毒性产品混合物中的化合物成为可能，只要它们具有不同的诱导动力学。
A new test to detect genotoxicity, that we refer to as the VITOTOX(R) test, was developed. Four gene fusions that are based on the Escherichia coli recN promoter were constructed and evaluated for their SOS response-dependent induction. The wild-type recN promoter, a derivative mutated in the second LexA binding site, a derivative with a mutated -35 region, and a derivative from which both the second LexA binding: site and the -35 region were mutated, were cloned upstream of the promoterless Vibrio fischeri luxCDABE operon of pMOL877, in such a way that lux became under transcriptional control of the recN promoter derivatives. The inducibility by the SOS response of the promoter constructs was tested in both E. coli and in the Ames test Salmonella typhimurium strains TA98, TA100 and TA104. In all strains, the highest sensitivity and induction was observed with the plasmids pMOL1067 and pMOL1068, that contain the lux operon under control of the recN promoter mutated in the second LexA binding site, or a recN promoter with a mutated -35 region, respectively. Therefore, strains containing pMOL1067 or pMOL1068 were further used for genotoxicity testing. With the VITOTOX test, genotoxicity was detected within 1-4 h. The VITOTOX test is very sensitive: for most products tested, the minimal detectable concentration (MDC) values were considerably lower (5 to > 100 times) than those described for the Ames test and the SOS chromotest. A good correlation was observed with the results from the Ames tests, but certain PAHs that are not mutagenic in the Ames test were genotoxic in the VITOTOX test. With the VITOTOX strains, the kinetics of SOS induction can be determined. This feature made it possible to distinguish between compounds in mixtures of genotoxic products so long as they had different induction kinetics.