Chemical stress sensitive luminescent human cells: molecular biology approach using inducible Drosophila melanogaster hsp22 promoter.
A whole-cell bioassay has been developed for the total toxicity testing of liquid samples. The method is based on the induction of the bioluminescent activity of genetically manipulated mammalian cells. For that purpose, transfection was used to introduce, in HeLa cells, a DNA sensing element that responds to chemical stress agents (heavy metals, genotoxic agents, and endocrine-disrupting chemicals). Such element was designed to direct the expression of a reporting gene (firefly luciferase) through the activation of Drosophila melanogaster hsp22 promoter. A molecular approach was conducted to optimize hsp22 promoter element in order to decrease the background expression level of the reporting gene and to increase the sensitivity of the bioassay for testing endocrine disruptors. As a result, in the presence of 20-100 microM cadmium chloride, a 6-fold increase in luciferase expression was obtained using a specially designed truncated hsp22 promoter construction. The following chemicals known to be found in the polluted samples were tested: CdCl2, Cd(NO3)2, NaAsO2, alachlore, fentine acetate, thiram, and maneb. The stressing effect of each of them was sensitively detected by the present bioassay in the 0.05-50 microM concentration range.
Mandon
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CA
Laboratoire de Génie Enzymatique et Biomoléculaire, UMR 5013 CNRS, Université Claude Bernard Lyon 1, 69 622 Villeurbanne cedex, France.
Diaz
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C
Arrigo
A-P
AP
Blum
L J
LJ
D000818:Animals; D001483:Base Sequence; D001681:Biological Assay; D019256:Cadmium Chloride / Q000494:pharmacology; D002470:Cell Survival; D002478:Cells, Cultured; D004305:Dose-Response Relationship, Drug; D029721:Drosophila Proteins / Q000235:genetics*; D004331:Drosophila melanogaster / Q000235:genetics*; D017353:Gene Deletion; D017930:Genes, Reporter; D005821:Genetic Techniques; D005822:Genetic Vectors; D006367:HeLa Cells; D006360:Heat-Shock Proteins / Q000235:genetics*; D006801:Humans; D008156:Luciferases / Q000378:metabolism; D008957:Models, Genetic; D008969:Molecular Sequence Data; D018384:Oxidative Stress; D011401:Promoter Regions, Genetic; D014162:Transfection
D029721:Drosophila Proteins; D006360:Heat-Shock Proteins; C490752:Hsp22 protein, Drosophila; D008156:Luciferases; D019256:Cadmium Chloride