Comparison of Ah receptor-mediated luciferase and ethoxyresorufin-O-deethylase induction in H4IIE cells: implications for their use as bioanalytical tools for the detection of polyhalogenated aromatic hydrocarbons.
A recombinant H4IIE rat hepatoma cell line (H4L1.1c4, H4IIE-luc), containing a luciferase reporter gene under control of dioxin-responsive enhancers, was examined for responsiveness to several polyhalogenated aromatic hydrocarbons (PHAHs). The recombinant cell system was compared with the widely used wild-type cell line (H4IIE-wt), which expresses Ah receptor-mediated cytochrome P450 1A induction. We also report an improved and down-scaled method for the H4IIE-wt bioassay which allows for the rapid screening of environmental samples for Ah-active PhAHs. This method employs 96-well plates, a plate-reading spectrofluorometer, and a fluorescence-based protein assay that enables the simultaneous measurement of resorufin and protein. Both cell lines demonstrated a dose-dependent increase in Ah receptor-mediated response upon exposure to a number of known Ah receptor agonists, including Halowax 1014. H4IIE-luc cells were 3-fold more sensitive than H4IIE-wt cells to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). The detection limit and ED50 for EROD induction by TCDD were 0.6 and 4.9 fmol/well (2,4 and 20 pM), respectively; for luciferase induction they were 0.2 and 1.4 fmol/well (0.8 and 5.6 pM). The detection limit for EROD induction in H4IIE-wt cells was a 50-fold improvement over that reported previously (Tillitt et al., Environ. Sci. Technol. 25, 87-92, 1991) and comparable to that of a chicken embryo primary hepatocyte bioassay (Kennedy et al., Anal. Biochem. 211, 102-112, 1993). The tested PHAHs exhibited a similar structure-activity relationship in H4IIE-luc as in H4IIE-wt cells. Binary mixtures of TCDD, PCB-126, and PCB-77 showed no departure from additivity in their combined responses when tested in H4IIE-wt cells. PCB-153 at the highest tested dose of 14 nmol/well (56 microM) significantly reduced the potency of TCDD and PCB-126 without affecting their efficacy in both H4IIE-wt and H4IIE-luc cells. These findings support the use of H4IIE-luc cells as an alternative bioanalytical tool to the wild-type cells for the detection of Ah agonists in environmental samples.
Sanderson
J T
JT
Department of Fisheries and Wildlife, Michigan State University, East Lansing 48824-1222, USA.
Aarts
J M
JM
Brouwer
A
A
Froese
K L
KL
Denison
M S
MS
Giesy
J P
JP
D000818:Animals; D019363:Cytochrome P-450 CYP1A1; D003577:Cytochrome P-450 Enzyme System / Q000096:biosynthesis*; D004790:Enzyme Induction / Q000187:drug effects; D005450:Fluorescamine; D008114:Liver Neoplasms, Experimental / Q000201:enzymology* / Q000473:pathology / Q000648:ultrastructure; D008156:Luciferases / Q000096:biosynthesis*; D010088:Oxidoreductases / Q000096:biosynthesis*; D011078:Polychlorinated Biphenyls / Q000032:analysis*; D000072317:Polychlorinated Dibenzodioxins / Q000032:analysis*; D051381:Rats; D018336:Receptors, Aryl Hydrocarbon / Q000502:physiology*; D012680:Sensitivity and Specificity; D012710:Serum Albumin, Bovine; D013329:Structure-Activity Relationship; D014407:Tumor Cells, Cultured
D000072317:Polychlorinated Dibenzodioxins; D018336:Receptors, Aryl Hydrocarbon; D012710:Serum Albumin, Bovine; D005450:Fluorescamine; D003577:Cytochrome P-450 Enzyme System; D011078:Polychlorinated Biphenyls; D010088:Oxidoreductases; D008156:Luciferases; D019363:Cytochrome P-450 CYP1A1