Process for inducing cytochrome p-450 enzymes in streptomyces bacteria and determining the mutagenicity of chemicals
申请人:E.I. DU PONT DE NEMOURS AND COMPANY
公开号:EP0347226A1
公开(公告)日:1989-12-20
A method of evaluating the potential mutagenic activity of a substance which comprises:
(a) forming a test culture, comprising a first culture medium, the substance to be evaluated, and cytochrome P-450 induced bacteria from the genus Streptomyces, which bacteria contain a plasmid which comprises a gene, which, upon exposure to a substance having mutagenic activity, confers to said bacteria the ability to grow in the presence of a preselected marker material;
(b) forming a control culture, comprising the cytochrome P-450 induced bacteria of step (a) and the culture medium of step (a) without the substance to be evaluated;
(c) separately harvesting the bacteria from the test culture and the control culture;
(d) separately culturing the harvested test bacteria and control bacteria under conditions which promote the formation of spores from said bacteria to form test spores and control spores;
(e) separately culturing the test spores and control spores in a second culture medium which (i) promotes the formation of bacteria from said spores and (ii) comprises the marker material;
(f) separately determining the fraction of the bacteria formed from the test spores and control spores which have the ability to grow in the presence of the preselected marker material, thereby determining a test revertant rate and a control revertant rate;
(g) determining the ratio of the test revertant rate to the control revertant rate; and
(h) determining that said substance has mutagenic activity whenever the ratio of step (g) is at least about 1.9.
一种评估物质潜在诱变活性的方法,包括
(a) 形成试验培养物,其中包括第一种培养基、待评估物质和链霉菌属的细胞色素 P-450 诱导细菌,该细菌含有质粒,该质粒包含一个基因,当暴露于具有致突变活性的物质时,该基因赋予所述细菌在预选标记物存在下生长的能力;
(b) 形成对照培养物,包括步骤(a)的细胞色素 P-450 诱导细菌和步骤(a)的培养基,其中不含待评估物质;
(c) 分别从试验培养物和对照培养物中收获细菌;
(d) 在促进所述细菌形成孢子的条件下,分别培养收获的试验细菌和对照细菌,以形成 试验孢子和对照孢子;
(e) 在第二种培养基中分别培养试验孢子和对照孢子,第二种培养基(i) 能促进所述孢子形成细菌,(ii) 包含标记材料;
(f) 分别确定由试验孢子和对照孢子形成的细菌中在预选标记材料存在下具有生长能力的部分,从而确定试验返祖率和对照返祖率;
(g) 确定试验逆转率与对照逆转率的比率;以及
(h) 只要步骤(g)中的比率至少为约 1.9,则确定所述物质具有诱变活性。