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), cont aining a luciferase reporter gene under control of dioxin-responsive e nhancers, was examined for responsiveness to several polyhalogenated a romatic 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 i mproved 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. H4 IIE-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), re spectively; 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 ce lls was a 50-fold improvement over that reported previously (Tillitt e t 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 st ructure-activity relationship in H4IIE-luc as in H4IIE-wt cells. Binar y mixtures of TCDD, PCB-126, and PCB-77 showed no departure from addit ivity in their combined responses when tested in H4IIE-wt cells. PCB-1 53 at the highest tested dose of 14 nmol/well (56 nM) significantly re duced 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-ty pe cells for the detection of Ah agonists in environmental samples. (C ) 1996 Academic Press, Inc.