BIOANALYSISVARIABLES THAT AFFECT TEQ DETERMINATIONS BY CALUX AND
GC-HRMS.
I. Windal1, I. Van Overmeire1, S. Carbonnelle1, N. Van Wouwe1, D. Brown2, G. Clark2, W.
Baeyens3, L. Goeyens 1
1

Scientific Institute of Public Health, rue J. Wytsman, 14,B-1050 Brussels Belgium
Xenobiotic Detection Systems Inc., 1601 East Geer Street, Suite S, Durham, NC 27704, USA
3
Free University of Brussels (VUB), Analytical Chemistry Laboratory, Pleinlaan, 2, B-1050 Brussels,Belgium
2Introduction
Recently, the CALUX assay (chemical-activated luciferase gene expression) has been proposed as ameans to conduct rapid analysis for dioxin and dioxin-like compounds. The genetically modified celllines used in the CALUX bio-assay respond to dioxin and dioxin-like compounds with the AhRdependant induction of firefly luciferase. However, the measure obtained by CALUX has to beconsidered with extreme caution, and everybody using CALUX results must be aware of theassumptions of the method and the importance of sample preparation. The objective of this paper is tofocus on parameters that may influence the CALUX measure.
In addition, chemical analysis and CALUX analysis are compared as tools for the evaluation of thetoxicity (expressed in TEQ) of a sample.
Substance specific effects
Possible Ah ligands
CALUX analysis will give a response for all compounds able to bind to and activate the Ahreceptor. These compounds have been divided into “classical” and “non-classical” Ah ligands byDenison & al.1. “Classical” Ah ligands are planar, aromatic and hydrophobic compounds, with astructure close to that of TCDD. Among them, we can cite PCDD/F, PCB, PBB, PBDE, PCT, PCN,PAH, aromatic amines, hexachlorobenzene. Some compounds with physical and structural properties
quite different from that of TCDD are also able to bind, in general weakly, to the Ah receptor. Thesecompounds were defined as “Non classical” Ah ligands. Among them, we can cite bilirubin, biliverdin,
tryptophan and its metabolites, some compounds from the benzimidazole group, corticosteroid groupor imidazolin group. This list is not exhaustive and research to identify AhR ligands continues. Whenperforming CALUX analysis “natural” ligands, e.g. bilirubin present in blood, should not contribute to
the response if the objective of the analysis is the determination of the toxicity due to pollutants.
Antagonism and synergy
The TEF principle used in chemical analysis assumes additive contribution of the different AhR
ligands. However, important nonadditive interactions between halogenated aromatic hydrocarbons
have been observed (see ref 2 for a review 2). These studies mainly focused on individual
polychlorinated biphenyls (PCB 52, 108, 153, 156, 159) or Aroclor 1254 which are able to inhibit thebiological response induced by TCDD. The high ratio of antagonist/agonist needed to observe theinhibition is usually observed in various environmental matrices. Note that the antagonism effectdepends on the cell lines used3. A synergistic interaction was also observed for corticostéroids:

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corticosteroids alone induce a weak response in CALUX, but dramatically enhance the response ofTCDD 4.
Procedural effects
When performing a CALUX analysis, the analysts have to choose parameters for the extraction,clean-up and analysis. These choices may have a considerable effect on the results obtained.
Extraction
The extraction determines which AhR ligands are extracted. Hard extraction conditions can lead tocomplete extraction of some compounds, but also to degradation of some other compounds. On theother hand, soft extraction conditions may lead to only partial recovery of analytes.
Clean-up
The clean-up step determines which compounds are eliminated from the extract and which
compounds are delivered to the cell. The effects of clean-up can be divided into two classes:• A decrease of the response in CALUX due to the elimination of some agonists. One example
could be a compound that gives a response in CALUX, but is destroyed during passage over acid silica.
Therefore if the extract is treated with acid silica this agonist from the sample does not contribute to theCALUX response.
• A change of the response in CALUX due to the suppression of some antagonistic or synergistic
effects. For example, some PCB’s do not give any response in the analysis when exposed alone, but
their presence in the extract decreases the response of PCDD/F. Consequently, if these PCB’s areeliminated from the extract (with a carbon column for example), the response due to the same quantityof PCDD/F in the extract may increase up to a factor of 2 in some cases.
Calculation of the percentage recovery
As no internal standard can be used, the percent recovery may only be estimated. This estimation
can be done via the addition of radiolabelled 14C 2,3,7,8 TCDD to a surrogate recovery sample (samematrix) subjected to the same extraction and clean-up. However, the percent recovery is determined fora different sample than the sample analyzed by CALUX. This implies that the sample preparation
should be very reproducible so that the difference between the percent recoveries of the 2 samples issmall. Besides this, the percent recovery is estimated only for the 2,3,7,8 TCDD and the percent
recovery of other compounds may be quite different. Another possibility is to calculate the recovery
from the TEQ level in a quality control sample around the limit of interest.
Analysis
The first parameter that influences the analysis itself is the cell line. Indeed, the structure of the Ah
receptor varies among species and consequently, the affinity of compounds for the Ah receptor varies
among species. The permeability of membranes, the AhR level, the AhR nuclear translocator protein
levels and/or other transcription factors, the antagonism or synergism effect of some compounds arealso species and tissue specific. Therefore the same mixture of AhR ligands could give a different
response when exposed in the same conditions to different cell lines 5.
The second parameter is the type of vehicle used to deliver the extract to the cells. According toSanderson et al. 6,dimethyl sulfoxide is more effective than isooctane in delivering the compounds tothe cells, resulting in several fold higher response.
The third parameter is the duration of exposure of the extract to the cells. Some compounds, like
PAH or aromatic amines are rapidly metabolized. The response to these compounds can only be390

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measured after a short incubation (several hours). Following longer exposures the response to thesecompounds is reduced as they are metabolized.7
Data interpretation
When analyzing an unknown sample by the CALUX assay, a calibration curve is established withTCDD standard solutions (response versus log concentration of TCDD) and the best equation fitting
the curve is used for calculation of results. The concentration of the sample, expressed as TEQ/sample,
is deduced by solving this equation with the response of the sample. However, this approach assumes
that the extract analyzed behaves like a diluted or concentrated solution of the standard. This implies
that the dose-response curves of the sample and of the standard are parallel and that the maximalachievable response (efficacy) for the standard and sample are identical. In practice, these assumptions
are usually not verified, and the concentration measured in the sample is a function of dose. The degreeof variation of the measure in function of dose is dependent of the degree of variation between theslopes of the standard and sample dose-response curves 8,9,. In order to limit these possible problems,
the acceptable responses in CALUX can be set to less than ½ the maximal response for the TCDD
standard curve.

CALUX versus chemical analysis for the evaluation of the toxicity of a sample
The main differences between CALUX and chemical analysis are summarized in the table.
CALUX
Analysis of all AhR ligands
in one rapid and inexpensive
analysis

Chemical analysis
Analysis only of thecompounds of interest. Timeconsuming and expensive if allAhR ligands are to be analyzed

Robustness

Sample preparation greatly
influences the measure

As long as there is nointerferences in GC-HRMS,
the sample preparation has noinfluence on the measure

Antagonism and synergism

Taken into account (in part)Not taken into accountTEF-REP principle

CALUX-REP lead to a direct
measurement based on cellularmechanism of toxicity

WHO-TEF estimated only forsome compounds on the basisof several biological tests

Precision

RSD associated with themeasure is quite high

RSD associated with theconcentration of a congener isquite low. But many
compounds have to be determined.

Detection limits

Very sensitive. Contribution ofall AhR ligands, even at very
low levels

Less sensitive. When somecompounds are under detection
limits, their contributions tothe total TEQ are ignored orSelectivity

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Additional information

estimated
Pattern of contamination,
useful for the identification ofpollution sources

Chemical analyses are usually taken as the reference method, given the actual measurement ofindividual congeners of PCDD/F in the sample. CALUX analyses are usually considered as a screening
approach, resulting in only an approximation of the toxicity, with a high RSD. However we have to beaware that even if the measure of the concentration of PCDD/F by chemical analysis is more precise,the result expressed as TEQ is also an estimation of the toxicity of the sample since 1) not allcompounds with dioxin-like properties are analyzed, 2) the TEF, if determined, is also an estimation 3)
non additive interactions are not taken into account 4) not detected compounds do not contribute to thetotal TEQ or their contribution is estimated.
Conclusions
Sample preparation greatly influences CALUX measurements. Standardization is needed to allow
comparison of results obtained in different laboratories.
CALUX and chemical analysis give different information, and are complementary: CALUX is avery powerful method to rapidly evaluate the toxicity of a sample due to dioxin-like compounds andmay be employed as a rapid screening for large numbers of samples. This allows for the screening ofsamples with respect to norms, comparison of sample contamination along a gradient or comparisonsof different populations. Chemical analysis gives information on the patterns of contamination, which
is valuable for identification of contamination sources and a precise measure of compounds that aresubject to regulation. However, it is important to emphasize that both results are only an approximationof the actual toxicity of a sample.
References
1. Denison, M.S.; Heath-Pagliuso, S. Bul.l Environ. Contam. Toxicol. 1998, 61, 557-568.
2. Safe, S. Teratogenesis, Carcinogenesis, and Mutagenesis 1997, 17, 285-304.
3. Aarts, J.M.M.J.G.; Denison, M.S.; Cox, M.A.; Schalk, M.A.C.; Garrison, P.M.; Tullis, K.; de Haan,L.H.J.; Brouwer, A. European Journal of Pharmacology Environmental Toxicology andPharmacology Section 1995, 293, 463-474.
4. Hoogenboom, L.A.P.; Hamers, A.R.M.; Bovee, T.F.H. Analyst 1999, 124, 79-85.
5. Garrison, P.M.; Tullis, K.; Aarts, J.M.M.J.G.; Brouwer, A.; Giesy, J.P.; Denison, M.S. Fundamental
and Applied Toxicology 1996, 30, 194-203.
6. Sanderson, J.T.; Aarts, J.M.M.J.G.; Brouwer, A.; Froese, K.L.; Denison, M.S.; Giesy, J.P. Toxicology
and Applied Pharmacology 1996, 137, 316-325.
7. Postlind, H.; Vu, T.P.; Tukey, R.H.; Quattrochi, C. Toxicol. App. Pharmacol. 1993, 118, 255
8. Villeneuve, D.L.; Blankenship, A.L.; Giesy, J.P. Environ. Toxicol. Chem. 2000, 19, 2835-2843.
9. Birnbaum, L. Environmental Health Perspectives 1994, 102, 157-167.

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