Comparison of Two Cell-Based Assays for Screening Dioxin and Dioxin-Like Compounds in Sediments

Technical Note DOER-C8

July 1999

Comparison of Two Cell-Based Assays

for Screening Dioxin and Dioxin-Like

Compounds in Sediments

PURPOSE: The purpose of this technical note is to report the results of studies comparing two

cell-based assays as screening tools for dioxins and related compounds in sediments. The Reuber

H4IIE rat hepatoma cell line and the recombinant 101L human hepatoma cell line were used to

assay 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) toxic equivalents (TEQs) in split samples of the

same sediment extracts. The two methods were compared for sensitivity and correlation with

analytical chemistry results. An interlaboratory comparison of results obtained with the 101L cells

was performed.

BACKGROUND: The reader is referred to DOER-C1, "Guidance for Performance of the H4IIE

Dioxin Screening Assay" (McFarland et al. 1998),1 for background. In that technical note, the use

of cultured mammalian cells as the basis of assays for dioxins in environmental samples was

described. The rationale for their use was given, and a detailed laboratory protocol for using the

Reuber H4IIE cell line for this purpose was provided. Herein, further studies are reported in which

a recombinant cell line, 101L, is used in side-by-side assays with the H4IIE on the same sediment

samples.

The basic mechanism that responds to the presence of polychlorinated dioxins/furans

(PCDD/PCDF), coplanar polychlorinated biphenyls (PCBs), and polynuclear aromatic hydrocar-

bons (PAHs) is the same in both H4IIE and 101L cells. The initial reaction is binding of the chemical

to a cytosolic receptor protein known as the aryl hydrocarbon receptor (AhR). This association

leads to the formation of a stable AhR-ligand complex that translocates to the cell nucleus and binds

with dioxin recognition elements (DREs) on the DNA. As a result, certain genes (e.g., CYP1A1)

are expressed, and detoxifying enzymes are synthesized. The amount of enzyme produced is directly

proportional to the concentration of bound chemical. Hence, the quantitation of one of these

enzymes serves as a measure of dioxin activity. In the assay using H4IIE cells, ethoxyresorufin-O-

deethylase (EROD) cleaves resorufin as a by-product that can be measured spectrofluorometrically.

Recently, advances in transgenic research have produced new ways of detecting dioxins and other

chemicals that bind with the AhR. Recombinant cell lines have been developed in which nonmam-

malian reporter genes are inserted downstream from the DREs in the DNA of the cells. When the

DREs are activated, the reporter gene is switched on, producing some protein other than EROD (or

another endogenous enzyme) that can be detected instrumentally. For example, the 101L cell line

is derived from human hepatoma HepG2 cells and is stably transfected with a plasmid containing

the human CYP1A1 promoter sequence fused to the firefly luciferase gene as a reporter (Anderson

et al. 1995). The induction of CYP1A1 results in the production of luciferase. Light produced by

the action of luciferase on the luciferin substrate can then be measured with a luminometer. The

P450 Reporter Gene System (P450RGS) is an assay based on 101L cells that is currently being used