CALUXTM RESULTS CORRELATE WITH GC/MS DATA FROMKAZAKSTAN BREAST MILK SAMPLES AND SUPPORT NEW TEF VALUESKim Hooper1, Douglas Hayward2, Michael Chu3, Meredith Anderson1 , William Farland4, George
Lucier5, and George Clark2
1Hazardous Materials Lab, California Environmental Protection Agency, Berkeley, CA 04704
US Food and Drug Administration, Washington, DC 20204
3
Xenobiotic Detection Systems, Inc., Durham, NC 27704
4
US Environmental Protection Agency, Washington, DC 20014
5
US National Institute of Environmental Health Sciences, Research Triangle Park, NC 27709
2Introduction
In 1994, in the first comprehensive investigation of persistent organochlorine contaminants in acountry of the former Soviet Union, we measured congener-specific levels of polychlorinatedbiphenyls (PCBs), polychlorinated dibenzo-p-dioxins (PCDDs), and polychlorinated dibenzofurans(PCDFs), as well as 19 organochlorine pesticides (OC) in breast milk samples collected using theWHO protocol from first-time mothers (“primiparae”) living in southern Kazakhstan (1-3). Highlevels (up to 80 pg/g fat) of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) were found in breast milk
samples from villages in a cotton-growing region in southern Kazakhstan, with TCDD contributing
70-80% of the I-TEQ (1-2). A follow-up study in 1997 measured levels of PCDDs/PCDFs insamples from the region’s cotton-growing State Farms (4). As part of our continuing
characterization of this TCDD contamination, we investigated the effectiveness of the CALUXTM
assay to screen and identify breast milk samples with high contaminant levels. In this case, the ITEQ arises from only two congeners, TCDD and the pentachlorodibenzo-p-dioxin. We report theresults of correlation studies on CALUXTM and GC/MS data using these samples..
Materials and Methods
Study design. Twenty-one breast milk samples and their organic extracts were used in thecorrelation studies. The samples were collected in 1997 using the WHO/EURO (5), protocol forbreast milk sample collection, in which donors were healthy primiparae with healthy infants 2-8weeks of age. The procedures followed in these exposure assessment studies (design, exposure
assessment questionnaire, informed consent, PCDD/PCDF target analytes, analytical methods, andstatistical analysis) are described in detail elsewhere (1-2).
Split samples of breast milk were analyzed by HRGCMS and by the CALUXTM assay. Results fromthe two methods were compared using statistical tests.
Analytical methods. Breast milk samples (100 mL) were collected from donors inchemically clean sample jars with teflon-lined caps, frozen immediately, and stored at -20°C until
analysis. Samples were thawed, shaken, and weighed, and a 5 gram aliquot was removed for theCALUXTM assay (“milk aliquot”). The remainder was extracted in ethanol:hexane:diethyl ether(2:1:1), and a 10% aliquot was set aside for CALUXTM assay (“extract aliquot”). The remainder ofthe extract was evaporated to dryness and the residue analyzed by HRGCMS
HRGCMS. Extracts of breast milk samples were spiked with 15 13C12-labeled
PCDD/PCDF standards and analyzed for congener-specific PCDD/PCDFs in a U.S. Food and Drug1

Administration (USFDA) laboratory using quadrupole ion storage tandem MS (7). Lipids were
determined gravimetrically, and residue levels were expressed as pg/g milk lipid. I-TEQs for
PCDDs/PCDFs were calculated using both old and new TEF values (8,9).
CALUXTM Assay. Xenobiotic Detection Systems, Inc. (XDS), has a patented
genetically engineered cell line which contains the firefly luciferase gene under trans-activational
control of the aryl hydrocarbon receptor (10). The cell line can be used for the detection and relative
quantification of PCDDs, PCDFs, and coplanar PCBs when used with our patent pending sample
processing procedure (11). The assay using this cell line is called the Chemical-Activated
Luciferase expression, or CALUXTM assay.
Using the CALUXTM assay, breast milk samples (“milk aliquots”) and the set of organic extracts(“milk extracts”), stored at –20° C, were analyzed for total TEQ activity. The “milk aliquots” (5 ml)
were transferred to hexane-rinsed glass vials with a PTFE-lined cap and were shaken and extracted
three times with a acetone/hexane mixture. The three extracts were pooled and evaporated todryness under nitrogen, and the remaining residue was weighed to determine organic extractables(“milk lipid”). The residues of “milk aliquots” and “milk extracts” were re-suspended in hexane andcleaned up for the bioassay using our proprietary patent pending clean-up procedure (11).
The sample extracts in DMSO were suspended in cell culture medium, just prior to dosing on
monolayers of H1.L1.6 mouse hepatoma cells that were grown in 96-well culture plates. In addition
to the dilution of samples, a standard curve of TCDD-concentrations was assayed (128.8, 64.4, 32.2,16.1, 8.0, 4.0, 2.0, 1.0, 0.5, and 0.1 ppt of TCDD). The plates were incubated for optimal inductionof luciferase activity in a humidified CO2 incubator. After the incubation, the media was removed
and the cells were microscopically observed for viability. The luciferase activity was quantified
using the substrate kit of Promega.
Statistical Analysis. Analytical data were stored in EXCEL 5.0 (Microsoft, Redmond,WA). All statistical analyses were conducted in STATA 5.0 (Stata Corp, College Station, TX).
Spearman Rank Correlation was used to assess the similarity of results from the HRGCMS andCALUXTM analytical methods. In addition, qualities of the distributions of the I-TEQ levels fromboth methods were compared using the Wilcoxon Signed Rank Test.
Results and Discussion
From the GC/MS analysis of milk samples, the ITEQs (pg/g fat) were calculated using either the oldTMor new TEFs (PeCDD TEFs of 0.5 vs 1.0, respectively). The CALUX assay was run on theprovided milk (n = 13) or milk extract samples (n=21). For the 8 extract samples with no milk
TMTMsamples, CALUX results were predicted from regression analysis of CALUX data from the 13TMmilk and milk extract samples. Thus, a CALUX data set was assembled on milk samples of 21values, 13 measured and 8 extrapolated.
TMCorrelation Analysis. The relationship between the GC/MS and CALUX results wasexamined by an extensive correlation analysis. The linear relationship between GC/MS andTM
CALUX results was stronger when new, rather than old, TEF values were used, and when all
PCDD/PCDF congeners were included in the calculation o f the TEQ (Table 1).

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Table 1: Comparison of GC/MS and CALUX
CALUX
Milk 13Milk(origina + 8l n=13) predicted(n=21)

GC/MS

ITEQ new TEF (n=21)
ITEQ old TEF (n=21)
TCDD+PeCDD new TEF (n=21)
TCDD+PeCDD old TEF (n=21)
TCDD (n=21)0.74180.68230.73630.66480.59420.78190.73790.75320.6857
0.6329Sensitivity, Specificity, Predictive Value of the CALUXTM assay. Based upon the distributional
characteristics of CALUX results for the 21 milk samples (13 measured and 8 predicted), four cutoff
points were selected for analysis of specificity, sensitivity, and predictive value of the CALUXTM
assay: 25, 38.12 (CALUXTM median ITEQ), 45, and 60 pg/g fat. Results are summarized in Table
2. The median cutoff gives the best balance of specificity and sensitivity.
Table 2: Sensitivity, Specificity, Predictive Value of CALUX Assay
Cutoff Point25 pg/g fat 38.12 pg/g 45 pg/g fat 60 pg/g fat
Estimate of Measurefat(CALUXmedian)Sensitivity
0.94
0.78
0.50
0.33
Specificity
0.20
0.75
0.92
1.00
Predictive Value (positive test result)
0.79
0.70
0.80
1.00
Predictive Value (negative test result)
0.50
0.82
0.75
0.79
Definitions:
Sensitivity: P(CALUX>=cutoff | GCMS>=cutoff)
Specificity: P(CALUX<cutoff | GCMS<cutoff)
Predictive value (positive test result): P(GCMS>=cutoff | CALUX>=cutoff)
Predictive value (negative test result): P(GCMS<cutoff | CALUX<cutoff)

Acknowledgements
We express our appreciation to the women of Kazakhstan who volunteered for this study. This study
was funded in part by the Trout Farm Ltd., the National Institute of Environmental Health Sciences,and the US Environmental Protection Agency.
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