Protocols for a Rapid Clean-up/Extraction Procedure and an Improved P450RGS Dioxin Screening Assay for Sediments

ERDC TN-DOER-C10

March 2000

Protocols for a Rapid Clean-up/Extraction

Procedure and an Improved P450RGS

Dioxin Screening Assay for Sediments

PURPOSE: The purpose of this technical note is to describe protocols for performance of an

improved biomarker-based screening assay for dioxin toxic equivalents (TCDD TEQs) in sediments

and other environmental samples using a recombinant human hepatoma cell line.

BACKGROUND: For background refer to McFarland, McCant, and Inouye (1998) in which the

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

described. There are several reasons why using cultured animal cells in assays for these contami-

nants is attractive. First, cell-based assays cost far less to perform and are done far more quickly

than, for example, gas chromatograph/mass spectrometer (GC/MS) analysis of dioxins; the differ-

ence in cost is at least an order of magnitude, and may be more depending on the number of samples

done in a batch, the number of replicates of each sample, and other factors. Additionally, the

sensitivity of the cell-based assays can be made to be of the same order as that of the GC/MS. For

these reasons, the cells can provide screening tests of sediment samples that permit the prioritization

of sites to receive definitive chemical analysis when analyses are required for regulatory or other

purposes. When cleanup and remediation involving soils or sediments contaminated with polyaro-

matic hydrocarbons (PAHs), polychlorinated biphenyls (PCBs), or polychlorinated dibenzodiox-

ins/furans (PCDDs/ PCDFs) are issues, the cells offer a less expensive alternative to chemistry for

monitoring the effectiveness of treatments. Since the costs are relatively much lower, many more

samples can be assayed for the same resource expenditure, allowing better characterization of the

extent of contamination at sites.

A second type of advantage involves the requirements of risk assessment. The mechanism by which

cell-based assays detect and measure the activity of dioxin and dioxinlike chemicals employs the

same cellular machinery responsible for the toxicity of these chemicals in whole organisms. While

the cell line responds to all dioxins/furans containing chlorines at the 2-, 3-, 7-, and 8-ring positions,

coplanar PCBs, and many of the PAHs, the most potent compound is 2,3,7,8- tetrachlorodibenzo-

p-dioxin (TCDD). Other dioxins and furans give lesser responses (from about 0.5 to only about

0.001 the response of TCDD), and the response to PCBs is even lower (0.008 to 0.000001 the

response of TCDD). The PAHs have the least potency as individuals, but because they are often

far more abundant than dioxins or PCBs, their contribution to the TCDD TEQ measured by the cells

can be significant. If both planar chlorinated hydrocarbons (dioxins, etc.) and PAHs are present,

the PAHs are easily removed during sample preparation. Unlike chemical analysis, cell-based assays

possess the ability to integrate the toxicological potency of complex mixtures of chemicals that have

similar modes of action but differ in potency as individuals.

Since the Reuber H4IIE rat hepatoma cell line was introduced in an assay for screening planar

chlorinated hydrocarbons in environmental samples (Bradlaw and Casterline 1979), a variety of cell

lines have been used in biomarker tests of exposure to chemicals and in identifying toxic substances

in environmental samples according to the primary physiological effect they produce. The most