APPLICATION OF THE CALUX BIOASSAY FOR THE DETERMINATION OF LOWTEQ VALUES IN MILK SAMPLES
Ilse Van Overmeire,1 Michael Chu,2 David Brown,2George Clark,2 Sophie Carbonnelle,1 and Leo Goeyens112Scientific Institute of Public Health, Juliette Wytsmanstraat 14, 1050 Brussels, Belgium
Xenobiotic Detection Systems, Inc., 1601 E. Geer Street, Suite S., Durham, NC 27704 USA

Introduction
The CALUX method has been evaluated earlier on its applicability as screening assay for food matrices.1 Severalsamples of largely different matrices and variable contamination levels (ranging from 0.5 to > 1000 pg TEQ/g lipid)
were analyzed by GC-HRMS and by the CALUX assay in order to compare and correlate obtained results.
This preliminary comparison has now been extended by new analyses of milk samples with low-level TEQ values.
The objective was to qualitatively and quantitatively screen those samples, taking into account the compulsory normof 5 pg TEQ/g lipid. Table 1 presents existing regulation for food items in Belgium.
Table 1Food matrix
Milk, bovine- and chicken meat, eggs, animal fats/oils+ derived products (> 2 % fat)Pork meat+ derived products (> 2 % fat)

Limit5 pg WHO-(PCDD/F)-TEQ/glipid
3 pg WHO-(PCDD/F)-TEQ/glipid

Concentrations in milk that were previously determined by GC-HRMS rarely exceed 5 pg TEQ/g lipid. These values
were obtained within the framework of a monitoring program launched in 1990.
The aim of this study was to investigate whether comparable results were obtained for poorly contaminated milk
samples (background levels). Therefore a number of samples, already analyzed by GC-HRMS, were reanalyzed byCALUX.

Materials and Methods
Samples
Samples were taken at the point of transport from farm to factory (mixed milk, MM) or at individual farms (farmmilk, FM). TEQ values, determined by GC-HRMS, were calculated according to WHO-TEF values and are basedon lower bound detection.
Sample preparation
The milk samples (60 g) were transferred to hexane-rinsed glass Erlenmeyers with PTFE-lined caps and wereshaken and extracted 4 times with an acetone/hexane mixture. The pooled extracts were evaporated under nitrogen,and the dried residues were weighed to determine organic extractables (“milk lipid”). The dried extracts were furthercleaned up in combination with PCDD/F isolation using a patent pending clean-up procedure.2

CALUX assay
In the CALUX assay a genetically engineered cell line is used, which contains the firefly luciferase gene undertrans-activational control of the aryl hydrocarbon receptor.3
The purified sample extracts in DMSO were suspended in cell culture medium prior to dosing monolayers of
H1.L1.6 mouse hepatoma cells that were grown in 96-well culture plates. In addition to the samples, a 2,3,7,8TCDD standard curve was generated on each plate. The plates were incubated for optimal induction of luciferase
activity in a humidified CO2 incubator. After incubation, the medium was removed and the cells were examined
microscopically for viability. The induced luciferase activity was quantified using the luciferase assay kit fromPromega.
Data and statistical analysis
The data for the 2,3,7,8-TCDD standard curve were fit to a sigmoid curve described by the Hill equation using leastsquares best fit modeling and responses of unknown samples were calculated as TCDD-TEQs by interpolation.
The Spearman rank correlation coefficient was calculated in STATISTICA.

Results and discussion
Correlation between CALUX and GC-MS results
8 Milk samples with dioxin levels between 1 and 6 pg TEQ/g lipid, as determined by GC-HRMS, were analyzed in
the CALUX assay. The correlation between the results obtained by CALUX and by GC-HRMS is shown in Figure
1.
Figure 1CALUX TEQ (pg/g lipid)

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56GC-MS PCDD/F-TEQ (pg TEQ/g lipid)

The Spearman rank correlation coefficient was found to be 0.94 (p = 0.0048) indicating a good correlation between
the results obtained by both techniques.
This correlation is in good agreement with results from a previous study using CALUX for the determination ofTEQ levels in breast milk.4 The TEQ values ranged from 25-60 pg TEQ/g lipid, what is significantly higher than theresults of our study.
Differences between chemo- and bio-analytical techniques
A few aspects must be considered when GC-MS and CALUX results are compared:•It has been demonstrated that CALUX-REPs differ from the WHO-TEFs.5,6•Chemo-analysis focuses on a limited number of dioxins (17 congeners with assigned TEFs). The CALUX
assay, however, detects all Ah agonists, such as other chlorinated and brominated congeners.
Implications for using the bioassay as a screening method

The CALUX assay is a promising tool for sample screening. A first objective of reference labs is to provide
statistical evidence for levels exceeding the compulsory norm concentration. Secondly, the technology could be usedfor quantitative determination, requiring extensive validation effort.
Acknowledgements
Financial support by the Office for Scientific, Technological and Cultural Affairs and from the Belgian Ministry ofPublic Health is gratefully acknowledged.
References
1. Van Overmeire, I., Goeyens, L., Beernaert, H., Srebrnik, S., De Poorter, G., Baeyens, W., Clark, G., Chu, M.,
Chu, A., Chu, D., Morris, R., Brown, D. (2000). A comparative study of GC-HRMS and CALUXTM TEQ
determinations in food samples by the Belgium federal ministries of public health and agriculture. Organohal.
Comp. 45, 196-199.
2. Chu, M.D., Clark, G.C. Patent application submitted.
3. Denison, M.S., Brouwer, A., Clark, G.C. United States Patent # 5,854,010.
4. Hooper, K., Hayward, D., Chu, M., Anderson, M., Farland, W., Lucier, G., Clark, G. (2000). CALUXTM resultscorrelate with GC/MS/MS data from Kazakhstan breast milk samples. Organohal. Comp. 45, 236-239.
5. Brown, D., Chu, M.,Van Overmeire, I., Chu, A., Clark, G.C., this conference.
6. Bovee, T.F.H., Hoogenboom, L.A.P., Hamers, A.R.M., Traag, W.A., Zuidema, T., Aarts, J.M.M.J.G., Brouwer,A., Kuiper, H. (1998). Validation and use of the CALUX-bioassay for the determination of dioxins and PCBs inbovine milk. Food Add. Contam. 15, 863-875.