Enhancing the response of CALUX and CAFLUX cell bioassays for quantitative detection of dioxin-like compounds

Abstract

Reporter genes produce a protein product in transfected cells that can be easily measured in intact or lysed cells and they have been extensively used in numerous basic and applied research applications. Over the past 10 years, reporter gene assays have been widely accepted and used for analysis of 2,3,7,8-tetrachlorodibenzo-p-dioxin and related dioxin-like compounds in various types of matrices, such as biological, environmental, food and feed samples, given that high-resolution instrumental analysis techniques are impractical for large-scale screening analysis. The most sensitive cell-based reporter gene bioassay systems developed are the mechanism-based CALUX (Chemically Activated Luciferase Expression) and CAFLUX (Chemically Activated Fluorescent Expression) bioassays, which utilize recombinant cell lines containing stably transfected dioxin (AhR)-responsive firefly luciferase or enhanced green fluorescent protein (EGFP) reporter genes, respectively. While the current CALUX and CAFLUX bioassays are very sensitive, increasing their lower limit of sensitivity, magnitude of response and dynamic range for chemical detection would significantly increase their utility, particularly for those samples that contain low levels of dioxin-like HAHs (i.e., serum). In this study, we report that the addition of modulators of cell signaling pathways or modification of cell culture conditions results in significant improvement in the magnitude and overall responsiveness of the existing CALUX and CAFLUX cell bioassays.

Figure 1

Temperature-dependent enhancement of TCDD-inducible reporter gene expression in mouse CAFLUX (H1G1.1c3) and CALUX (H1L6.1c2) cell lines. H1L6.1c2 and H1G1.1c3 cells were incubated at the indicated temperature with increasing concentrations of TCDD for 24 h. EGFP and luciferase activity were determined as described. Values are expressed as the relative light or fluorescence units (RLUs and RFUs, respectively) and represent the mean ± SD of triplicate determinations.

Figure 2

Temperature-dependent enhancement of reporter gene expression in mouse CALUX (H1L6.1c2) and CAFLUX (H1G1.1c3) cell lines by sediment extracts. H1L6.1c2 and H1G1.1c3 cells were incubated at the indicated temperature with either increasing concentrations of TCDD standard ((a) and (c)) or a sediment sample extract ((b) and (d)) for 24 h. Luciferase and EGFP activity were determined as described. Values are expressed as the relative light or fluorescence units (RLUs and RFUs, respectively) and represent the mean ± SD of triplicate determinations.

Figure 3

Addition of dexamethasone (DEX) synergistically increases TCDD-dependent induction of luciferase activity in the CALUX cell bioassay. H4L1.1c4, H1L1.1c2 and G16L1.1c8 cells were incubated with 1 nM TCDD, 10 μM DEX or TCDD plus DEX for 4 h as indicated. Luciferase activity was determined as described. Luciferase values are expressed as the percentage of that induced by 1 nM TCDD and represent the mean ± SD of triplicate determinations.

Figure 4

Coincubation with PMA synergistically increases TCDD-dependent induction of luciferase activity in the CALUX cell bioassay. Mouse CALUX (H1L1.1c2) cells were incubated with increasing concentrations of TCDD or beta-naphthoflavone (BNF) in the absence or presence of PMA (81 nM) for 4 h. Luciferase activity was determined as described under Materials and methods. Luciferase values are expressed as the percentage of that induced by 1 nM TCDD and represent the mean ± SD of triplicate determinations.

Figure 5

Dose dependent luciferase reporter gene expression induced by sediment sample extracts in mouse CALUX (H1L6.1c2) cells. H1L6.1c2 cells were incubated with the indicated equivalent concentration of sediment extract for 24 h. Luciferase activity was determined as described under Materials and methods. Values are expressed as the percentage of that induced by 1 nM TCDD and represent the mean ± SD of triplicate determinations.