Chemical-activated luciferase gene expression (CALUX): a novel in vitro bioassay for Ah receptor active compounds in sediments and pore water.
This study demonstrates that the novel in vitro CALUX (chemical-activated luciferase expression) assay is a rapid, sensitive assay for assessing the toxic potency of (mixtures of) aryl hydrocarbon receptor (AhR)-active compounds in sediments and pore waters. A rat hepatoma (H4IIE) cell line, stably transfected with a construct containing the dioxin-responsive element (DRE) sequence and the luciferase reporter gene, was used to determine the relative potency or the total activities of AhR-active compounds in sediment and pore water extracts. This novel CALUX assay had a detection limit of 0.5 fmol of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). The sensitivity and linear working range was slightly better than for the ethoxyresorufin O-deethylase (EROD) assay in H4IIE wild type cells. The primary improvement of the CALUX assay compared to the EROD assay, however, is that the CALUX assay is insensitive to substrate inhibition. The CALUX activity induced by organic extracts from 450-mg aliquots of sediment or 250-microl aliquots of pore water corresponded with the instrumentally analyzed degree of pollution of the sediment. Using pore water, only a simple and rapid extraction procedure was needed, without additional clean-up to prevent cell death. The response from pore water samples in an 8-day early life stage test with zebra fish (Branchydanio rerio) corresponded with the CALUX induction, although the correlation was sometimes disturbed by heavy metals. Two polychlorinated terphenyl mixtures, the PCB-substitute Ugilec 141, polybrominated diphenylethers, and the PCB-mixture Clophen A50 were tested in the CALUX assay and had induction potencies that were 10(-4)-10(-7) compared to TCDD.
Murk
A J
AJ
Department of Toxicology, Agricultural University Wageningen, The Netherlands.
Legler
J
J
Denison
M S
MS
Giesy
J P
JP
van de Guchte
C
C
Brouwer
A
A
D000818:Animals; D001681:Biological Assay; D006528:Carcinoma, Hepatocellular / Q000201:enzymology; D019363:Cytochrome P-450 CYP1A1 / Q000037:antagonists & inhibitors; D004305:Dose-Response Relationship, Drug; D004784:Environmental Monitoring / Q000379:methods*; D004785:Environmental Pollutants / Q000032:analysis / Q000633:toxicity; D015870:Gene Expression / Q000187:drug effects*; D019015:Geologic Sediments / Q000737:chemistry*; D006846:Hydrocarbons, Halogenated / Q000032:analysis / Q000633:toxicity*; D008156:Luciferases / Q000096:biosynthesis / Q000235:genetics*; D051379:Mice; D011078:Polychlorinated Biphenyls / Q000633:toxicity; D000072317:Polychlorinated Dibenzodioxins / Q000633:toxicity; D051381:Rats; D018336:Receptors, Aryl Hydrocarbon / Q000378:metabolism*; D015203:Reproducibility of Results; D018570:Risk Assessment; D012623:Seawater / Q000737:chemistry*; D014407:Tumor Cells, Cultured; D015027:Zebrafish / Q000502:physiology
D004785:Environmental Pollutants; D006846:Hydrocarbons, Halogenated; D000072317:Polychlorinated Dibenzodioxins; D018336:Receptors, Aryl Hydrocarbon; D011078:Polychlorinated Biphenyls; D008156:Luciferases; D019363:Cytochrome P-450 CYP1A1