DRESSA: biosensing of dioxin and dioxin-like chemicals using secreted alkaline phosphatase.
In this article, we describe a highly sensitive biosensing system, DRESSA, for detection of dioxin and dioxin-like chemicals. Tandem copies of the dioxin-responsive element (DRE) fused to a minimal viral promoter were subcloned into an expression plasmid upstream of a secreted alkaline phosphatase (SEAP) gene. When murine hepatoma cell line Hepa-1c1c7 was stably transfected with this construct, established sensor clones secreted SEAP following stimulation with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). A clone HeDS49 was found to be extremely sensitive; it secreted SEAP in response to TCDD in dose- and time-dependent manners, and the minimal detection limit was 100 fM. To detect more than 6 pM of TCDD, the whole assay time (from cell seeding to measurement of SEAP activity) could be reduced to 4h. Secretion of SEAP was induced selectively by other activators of DRE (3-methylcholanthrene, benzo[a]pyrene, and beta-naphthoflavone) but not by activators of unrelated responsive elements. These data suggested that because of the rapidity, easiness, specificity, and high sensitivity of DRESSA, it is more suitable than currently available detection systems for dioxin and dioxin-like chemicals and would be of great advantage to high-throughput screening of these pollutants in environmental samples.
Kasai
Ayumi
A
Department of Biochemistry, Interdisciplinary Graduate School of Medicine and Engineering, University of Yamanashi, Tamaho, Yamanashi 409-3898, Japan.
Hiramatsu
Nobuhiko
N
Meng
Yiman
Y
Yao
Jian
J
Takeda
Masayuki
M
Maeda
Shuichiro
S
Kitamura
Masanori
M
D000469:Alkaline Phosphatase / Q000235:genetics / Q000378:metabolism*; D000818:Animals; D001564:Benzo(a)pyrene / Q000494:pharmacology; D006528:Carcinoma, Hepatocellular / Q000378:metabolism / Q000473:pathology; D019363:Cytochrome P-450 CYP1A1 / Q000235:genetics; D004147:Dioxins / Q000494:pharmacology*; D004790:Enzyme Induction / Q000187:drug effects; D005786:Gene Expression Regulation / Q000187:drug effects*; D008113:Liver Neoplasms / Q000378:metabolism / Q000473:pathology; D008324:Mammary Tumor Virus, Mouse / Q000235:genetics; D008748:Methylcholanthrene / Q000494:pharmacology; D051379:Mice; D010957:Plasmids; D000072317:Polychlorinated Dibenzodioxins / Q000494:pharmacology; D011401:Promoter Regions, Genetic / Q000235:genetics; D020218:Response Elements / Q000502:physiology*; D012680:Sensitivity and Specificity; D014162:Transfection; D014407:Tumor Cells, Cultured; D019324:beta-Naphthoflavone / Q000494:pharmacology
D004147:Dioxins; D000072317:Polychlorinated Dibenzodioxins; D001564:Benzo(a)pyrene; D008748:Methylcholanthrene; D019324:beta-Naphthoflavone; D019363:Cytochrome P-450 CYP1A1; D000469:Alkaline Phosphatase