Novel mammalian cell lines expressing reporter genes for the detection of environmental chemicals activating endogenous aryl hydrocarbon receptors (ArhR) or estrogen receptors (ER).
We have constructed two vector systems (pDMS5, pSAB2) containing the promoter regions of the human CYP1A1 gene including xenobiotic response elements or the promoter region of the Xenopus laevis vitellogenin A2 gene including estrogen response elements, respectively, and the genes for green fluorescent protein and firefly luciferase. These vectors were transfected into CHO-K1 cells. Transiently transfected cells consistently responded to 1 nmol/l TCDD (dioxin) or 10 nmol/l 17ss-estradiol, respectively, with a 3-5 fold increase in luciferase activity. Permanent cell lines were selected by culturing transiently transfected cells under continued presence of antibiotics and dilution cloning. Cells which had stably integrated the vector-DNA into the genomic DNA were selected. SiF6 cells responded to treatment with TCDD, PCB126, benzo(a)pyrene or indirubin-3'-monoxime in the concentration range between 0 and 1 micromol/l. SiG12 cells responded to treatment with bisphenol A, 4-MBC and DDT in the concentration range between 0 and 10 micromol/l. Compared with the controls, luciferase mRNA-abundance (semi-quantitative RT-PCR) and luciferase activity (luminescence assay) were elevated up to 3-fold. Resveratrol or tamoxifen, respectively, worked as full antagonists. Luciferase expression was increased upon treatment of cells with extracts of spiked soil samples indicating that our systems are suited for screening of environmental samples.
Minh
Si Do
SD
Animal Physiology and Biochemistry, Zoological Institute and Museum, Ernst Moritz Arndt-University, Johann Sebastian Bach-Strasse 11/12, D - 17487 Greifswald, Germany.
Below
Sabine
S
Müller
Christian
C
Hildebrandt
Jan-Peter
JP
D000818:Animals; D016466:CHO Cells; D002460:Cell Line; D006224:Cricetinae; D003412:Cricetulus; D019363:Cytochrome P-450 CYP1A1 / Q000235:genetics / Q000378:metabolism*; D004785:Environmental Pollutants / Q000032:analysis / Q000633:toxicity*; D015870:Gene Expression; D017930:Genes, Reporter / Q000235:genetics; D005822:Genetic Vectors / Q000378:metabolism; D006801:Humans; D008156:Luciferases / Q000187:drug effects / Q000235:genetics / Q000378:metabolism; D018336:Receptors, Aryl Hydrocarbon / Q000187:drug effects* / Q000378:metabolism; D011960:Receptors, Estrogen / Q000187:drug effects* / Q000378:metabolism; D020218:Response Elements / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014162:Transfection / Q000379:methods; D014819:Vitellogenins / Q000235:genetics; D015262:Xenobiotics / Q000032:analysis / Q000633:toxicity; D014982:Xenopus laevis
D004785:Environmental Pollutants; D018336:Receptors, Aryl Hydrocarbon; D011960:Receptors, Estrogen; D014819:Vitellogenins; D015262:Xenobiotics; D008156:Luciferases; D019363:Cytochrome P-450 CYP1A1