VALIDATION STUDY FOR THE USE OF THE DIOXIN RESPONSIVECALUXTM ASSAY FOR ANALYSIS OF JAPANESE ASH AND SOILSAMPLESDavid Brown1, Yasuyuki Kishimoto2, Osamu Ikeno2, Michael Chu1, Jun Nomura2, Toshiaki
Murakami2 and Hiroshi Murata2
1

Xenobiotic Detection Systems International, Inc., 1601 E. Geer Street, Suite S, Durham, NC
27704 USA
2
Hiyoshi Corporation, 908 Kitanoshocho, Omihachiman, Shiga, 523-8555, Japan

Introduction
In Japan incineration is a common method for disposing of municipal waste and it is estimated that
more than 10,000 incinerators of various capacities are currently in operation. In the past coupleof years there has been an increased concern regarding the emission from these incinerators andother the emissions of other industries. In particular the concern has focused on the inadvertent
production and release of chlorinate aromatic compounds such as polychlorinated biphenyls(PCBs), polychlorinated dibenzo-p-dioxins (PCDDs) and polychlorinated dibenzofurans (PCDFs).
In August 1997 the Japanese government addressed these concerns by amending the cabinet ordersof the Air Pollution Control Law and Waste Management and Public Cleansing Law. These
amendments implemented stricter regulations on incinerators and other industries that emit PCBs,
PCDDs and PCDFs1.
These amendments placed an immediate limit on emissions from new facilities that will emit thesecompounds and provided for gradually more strict limits for existing facilities over a five-yearperiod. In order to comply with these new limits it was expected that monitoring by chemical
analysis would have to increase. This raised concerns that the chemical analysis by HRGCMS forcompliance might be an economic hardship for some of the regulated industries and that theincreased demand might outstrip the capacity of the existing analytical laboratories. Based onthese concerns the Japanese government and private corporations began to examine the possibilityof using alternative testing methods to monitor for the presence of these compounds. In this article
we report the results from a preliminary validation study conducted by Hiyoshi Corporation andXenobiotic Detection Systems, Inc (XDS). This study used a blinded format to compare theresults from the dioxin responsive CALUXTM assay with HRGCMS data.
Materials and Methods
Study Design. Twenty-five ash and soil samples were split and given a code number.
The split samples were analyzed by HRGCMS and by the CALUXTM assay. The results fromthese two methods were sent to an independent statistician for analysis.
HRGCMS. Sediment and ash samples were spiked with 13C12-labeled PCDD/PCDF
standards and analyzed for congener-specific PCDD/PCDFs at Hiyoshi Corporation. I-TEQs for
PCDDs/PCDFs were calculated using TEF values2.

CALUXTM Assay. XDS has a patented genetically engineered cell line (mouse hepatomaH1L1) that contains the gene for firefly luciferase under transactivational control of the arylhydrocarbon receptor3. This cell line can be used for the detection and relative quantificatation ofa sample’s total dioxin I-TEQ. Using a patent pending sample processing procedure it is also
possible to use the CALUXTM assay to estimate the I-TEQ contributions of PCDDs/PCDFs or theI-TEQ contributions of the coplanar PCBs4. The assay that uses this cell line is called theChemically-Activated Luciferase Expression or CALUXTM assay.
The samples were extracted using a modification of the EPA 8290 extraction method5. Briefly, thedried samples were ground and one gram aliquots were placed in solvent cleaned glass vials withPTFE lined caps. The sample was extracted with a 20% solution of methanol in toluene then twicewith toluene. During each extraction step the samples were incubated in an ultrasonic water bath.
The three extracts from each sample were filtered, pooled and concentrated by vacuum
centrifugation. The sample extract was suspended in hexane and prepared for the bioassay by aproprietary clean up method. The eluate from the clean up method was concentrated undervacuum into dimethyl sulfoxide (DMSO). The DMSO solution was used to dose the geneticallyengineered cells in the CALUXTM assay.
Prior to dosing the cells, the sample extracts in DMSO were suspended in cell culture medium.
This medium was then used to expose monolayers of the H1L1 cell line grown in 96 well culture
plates. In addition to the samples, a standard curve of 2,3,7,8-tetrachlorodibenzo-p-dioxin(TCDD) was assayed (161, 80.5, 40.2, 20.1, 10.1, 5.0, 2.5, 1.2 and 0.6 parts per trillion (ppt)
TCDD). The plates were incubated for a time to produce optimal expression of the luciferase
activity in a humidified CO2 incubator. Following incubation, the medium was removed and thecells were examined microscopically for viability. The induction of luciferase activity wasquantified using the luciferase assay kit from Promega.
Results and Discussion
From the GC/MS analysis of the samples, the I-TEQs were calculated using the TEF values for theindividual congeners. The sample I-TEQs were estimated by the CALUXTM assay by comparing
the response of the sample extract to the standard curve for 2,3,7,8-TCDD.
The correlation coefficient between the results is acceptable, (r = 0.94), however, there wereseveral samples where the CALUXTM assay over-estimated the sample I-TEQ relative to theGC/MS results. These over estimates of sample I-TEQ by the CALUXTM assay were probably due
to the fact that the contribution of the coplanar PCB was included in the CALUXTM estimates
while the GC/MS estimates only include the contributions from the PCDDs and PCDFs. We planto test this hypothesis by reanalyzing these samples using our patent pending clean up process thatallows us to differentiate between the contributions of the coplanar PCBs and PCDDs/PCDFs.
Figure 1 provides a graphical comparison of the results for the 25 samples from GC/MS andCALUXTM assay. A diagonal line representing a 1 to 1 relationship has been drawn on the graphto help with comparisons. By examining the graph it is evident that there is a strong correlation
between the CALUXTM results and the GC/MS data. It is also evident that the CALUXTM I-TEQestimates tend to be higher than the GC/MS data (21 of 25 samples), especially for lower levelsamples. Part of this could be related to the point made above: as currently reported theCALUXTM I-TEQ includes the contribution from the coplanar PCBs while the GC/MS results do
not include the coplanar PCB contribution to I-TEQ. Over estimation by the CALUXTM assay is
not a significant concern. The CALUXTM assay is intended to be a screening assay that can beused to identify samples that need to be analyzed by more time consuming and expensive chemicalanalysis methods (GC/MS). In general it is better for a screening assay to provide a high estimate
as false positives are more acceptable than false negatives for a screening assay. Of the foursamples that were underestimated by the CALUXTM assay, the results for three were very similar to
the GC/MS results. One of the samples that was underestimated by the CALUXTM assay would beconsidered a false negative (1 of 25 or 4%). This sample will be reanalyzed to determine whether
the incidence of false negatives can be further reduced.
Figure 1.

Comparison of GC/MS and CALUX Results
100000ppt by CALUX

10000
10001001010.10.1110100100010000

ppt by GC/MSConclusions
The data presented in this report showed the usefulness of the biologically based CALUXTM as analternative method to GC/MS for estimating TEQ levels in soil and ash samples. The CALUXTM
assay is a rapid and cost effective method that showed good correlation with GC/MS results with aminimal number of false negatives. Based on these results the CALUXTM assay should be usefulas a screening assay for estimation of TEQ in ash and soil samples.
Acknowledgements
This study was funded in part by a grant from the National Institute of Environmental Health
Sciences.
References
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