ANA - Bioanalytical Approaches for POPs Detection
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Mathematical Model Predictions of GC/MS Data for Dioxin Contaminated Environmental
Samples Using CALUX® by XDS
George C. Clark1, Jean Orelien2, John Gordon1, Andrew C. Chu1, Michael D. Chu3, Michael S. Denison4
1

Xenobiotic Detection Systems, Inc.
SciMetrika, LLC.
3
Alta Analytical Perspectives
4
Department of Environmental Toxicology, Univ. of California
2Introduction
The CALUX® by XDS bioassay is a mechanistically based recombinant cell bioassay designed to be a rapidscreening tool to evaluate the level of polychlorinated dioxin-like contaminants (i.e., dibenzo-p-dioxins, dibenzofuransand biphenyls) present in sample extracts. While the methodological aspects of the bioassay system has beenstreamlined and optimized, the bioassay system is currently undergoing extensive validation using a wide variety ofmatrices. However, one aspect that is still evolving is the development and validation of mathematic approaches forthe accurate estimation of these chemicals from bioassay analysis. Development of an appropriate mathematic
model designed to predict GC/MS TEQ results from cell bioassay data would both improve and validate thecomparability of GC/MS and CALUX® by XDS methods for determination of polychlorinated dioxin/furan andbiphenyl concentrations from a Total equivalence perspective. Such a mathematical model was applied to thisanalysis.
Methods and Materials
Environmental samples for analysis. In March 2004, the U.S. EPA and the Battelle Corporation conducted arigorous cross validation SITE study comparing CALUX® by XDS screening estimates to high resolution gas
chromatography/mass spectrometry (GC/MS) analysis of chlorinated dioxins/furans and biphenyls in environmentalsamples. The SITE study consisted of 209 samples analyzed by both methods. For CALUX analysis, forty of thesesamples were analyzed in a mobile lab stationed in Saginaw, Michigan MI, while the remaining 169 were analyzed
on-site at XDS in North Carolina.
Sample preparation and CALUX® by XDS bioassay. The CALUX® by XDS bioassay is a reporter gene assay inwhich a mouse hepatoma (Hepa1c1c7) cell has been stably transfected with a vector that contains the luciferase
gene under transactivational control of the Ah receptor1-3. Combined with a patented sample processing system toreduce contamination with non-dioxin-like agonists for the Ah receptor, the CALUX® by XDS bioassay can be used
for estimation of TEQ contamination with dioxin-like chemicals of environmental, biological, and chemical samples46. In this study, a 0.5 g sub-sample of each soil sample or solution was extracted with toluene and processed throughour patented cleanup procedure4. The isolated extract was exchanged into dimethylsulfoxide and applied to 96 wellplate monolayers of our genetically engineered cells and incubated for maximal induction of the firefly luciferase
within the cells. Light produced by the firefly luciferase enzyme was measured with an assay kit from Promega andquantified on a Berthold Orion MicroplateLuminometer. Dilution analysis was performed for range finding of theapproximate concentration of the processed sample that induced maximal luciferase activity for quantification in theCALUX® by XDS bioassay. Dioxin-like TEQ activity was estimated from a standard curve of 2,3,7,8 tetrachlorodibenzo-p-dioxin using a four-parameter Hill equation as previously described6,7.
To model GC/MS as a function of CALUX® by XDS, we fitted a statistical model with generalized estimatingequation (GEE)7 that accounts for the correlation due to replicates (observations from the same sample) using abase 10 logarithm transformation of the two variables. Observations that contain no dioxins were removed from themodel. Similarly, observations that contain CALUX® by XDS values that would have been removed under standard
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ANA - Bioanalytical Approaches for POPs Detectiontest protocol were not included in the model.
Results and Discussion
These screening data points do not demonstrate a strict one to one correspondence as shown in Figure 1. This is
expected since the TEQ estimates for CALUX® by XDS receptor based technology provides an estimate ofreceptor-dependent activation of the Ah receptor by chemicals present in the extract and many biological responses
are logarithmically related to concentration. The plotting of this relationship generates one model that describes therelationship.

The deviation from a direct relationship occurs for a number of reasons including such factors as the presence ofother halogenated dioxins and furans and other Ah receptor agonists in the fractionated sample, differences in theREP values of XDS cells versus the TEF values used to scale the GC/MS estimates of TEQ, and the kinetics ofbinding and activation of the receptor. Modeling the data we can derive a formula to transform the CALUX® by XDS
data to provide a better estimate of the GC/MS data. The relationship between CALUX® by XDS and GCMS isgiven by the following equation:
LOG(GC/MS)=0.6093*LOG(CALUX)+0.0584*[LOG(CALUX)]2
Figure 2 (dots representing observed and the line representing the predicted) shows the relationship between
predicted and observed values from the model. Overall, the values for the predicted and observed are close to theidentity line indicating a good fit. For larger dioxin levels (greater than 375), the model tends to underestimate thetrue GC/MS value.

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ANA - Bioanalytical Approaches for POPs Detection

Figure 3 (dots representing observed and the line representing the predicted) shows fitted values from the modelagainst CALUX® by XDS values. The graph shows that the model overall is a good fit for values of CALUX® by XDS
less than 1,500. For values beyond 1,500, the model fits the data reasonably well although the value of GC/MS tends
to be estimated.

This modeling exercise shows how values of GC/MS can be predicted from CALUX® by XDS for dioxins. Several
limitations need to be noted. First, the model appears to underestimate the true GCMS values over some range ofthe data. Accordingly, there may be a need to refine the model using splines (that is by assuming for example thatthe slope of the line is not constant). Second, values beyond 1000 for GC/MS were excluded. Hence, care should be
exercised when using the model for samples that contain high concentration of dioxin/furans. When there is a highconcentration of dioxins in the sample (beyond 1,000), the CALUX® by XDS assay yields higher than usual values,
which would result in overestimation of the true GC/MS value. Thus, with the CALUX ® by XDS bioassay, there isunlikely to be a false negative result with a concentration of dioxin/furans beyond the regulatory threshold. Finally, themodel has not yet been determined as to whether it also applies to other dioxin-like chemicals such as thepolychlorinated biphenyls.
Acknowledgements: This research was supported generously by NIEHS SBIR grant ES08372-02 and a NIEHS
Superfund Basic Research Grant ES04699.

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2. Denison, M. S., Brouwer, A., and Clark, G. C. U.S. Patent No. 5,854,010, Dec. 29, 1998.
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4. Chu, M. D. and Clark, G. C. Patent No. 6,750,431, April 13, 2004.
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