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Environ Toxicol Chem. Author manuscript; available in PMC 2010 April 23.

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Published in final edited form as:
Environ Toxicol Chem. 2007 June ; 26(6): 1122–1129.

ANALYSIS OF DIOXINS IN CONTAMINATED SOILS WITH THE
CALUX AND CAFLUX BIOASSAYS, AN IMMUNOASSAY, AND
GAS CHROMATOGRAPHY/HIGH-RESOLUTION MASS
SPECTROMETRY
Malin Nording*,†,‡, Michael S. Denison§, David Baston§, Ylva Persson†, Erik Spinnel†, andPeter Haglund††Environmental Chemistry, Umeå University, SE-901 87 Umeå, Sweden‡Swedish Defense Research Agency, SE-901 82 Umeå, Sweden

NIH-PA Author Manuscript§Department of Environmental Toxicology, University of California, Davis, California 95616, USA

Abstract

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The chemically activated luciferase expression assay, the chemically activated fluorescence
expression assay, and the enzyme-linked immunosorbent assay (ELISA) are all bioanalyticalmethods that have been used for the detection and quantification of polychlorinated dibenzo-pdioxins and polychlorinated dibenzofurans (PCDD/Fs). However, no comparisons of the results
obtained by these three methods have been published analyzing identical replicates of purified
sample extracts. Therefore, we have evaluated the performance of each of these methods foranalyzing PCDD/Fs in aliquots of extracts from aged-contaminated soil samples and compared theresults with those obtained by gas chromatography/high-resolution mass spectrometry (GC/HRMS). The quantitative performance was assessed and the effects of sample purification anddata interpretation on the quality of the bioassay results were investigated. Results from thebioanalytical techniques were, in principle, not significantly different from each other or from the
GC/HRMS data (p = 0.05). Furthermore, properly used, all of the bioanalytical techniques
examined were found to be sufficiently sensitive, selective, and accurate to be used in connection
with soil remediation activities when aiming at the remediation goal recommended by the U.S.
Environmental Protection Agency (i.e., < 1,000 pg toxic equivalency/g). However, a site-specificcorrection factor should be applied with the use of the ELISA to account for differences between
the toxic equivalency factors and the ELISA cross-reactivities of the various PCDD/F congeners,
which otherwise might significantly underestimate the PCDD/F content.

Keywords
CALUX; CAFLUX; Enzyme-linked immunosorbent assay; Soil; Dioxin

INTRODUCTION
Dioxin is a generic term used for polychlorinated dibenzo-p-dioxins and polychlorinateddibenzofurans (PCDD/Fs) and it typically refers to the most potent or toxic PCDD/F© 2007 SETAC*To whom correspondence may be addressed (malin.nording@chem.umu.se).

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congener, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). The TCDD and other PCDD/F
congeners with chlorine substituents in the 2,3,7,8-positions can cause a wide spectrum of
biological and toxicological effects, including immunotoxicity, carcinogenicity, and adverse
effects on reproduction [1–3]. The aryl hydrocarbon receptor (AhR) plays a crucial role inthe mechanism mediating the toxic action of these compounds and it begins with the highaffinity binding of the ligand (PCDD/F) [4–6]. Subsequent translocation of the complex into
the nucleus of the cell, dimerization of the AhR with the AhR nuclear translocator (Arnt)
protein, and binding of the ligand:AhR:Arnt complex to its DNA recognition sequence leads
to increased transcription and translation of certain genes, including that of cytochrome
P4501A1 (CYP1A1). All dioxin-like compounds are assumed to act through this AhR signaltransduction pathway.

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Because the toxic response is mediated through a common mechanism, the toxicity of each
2,3,7,8-substituted PCDD/F congener can be compared to that of TCDD. For this purpose,
toxic equivalency factors (TEFs) have been assigned to seven PCDDs, ten PCDFs, and 12
polychlorinated biphenyls (PCBs), that all meet the basic TEF criteria (i.e., they are
persistent in the environment, structurally related to dioxins, exert their action through the
AhR, and produce a similar spectrum of toxic and biological responses) [7,8]. The PCBs ofinterest might adopt a planar configuration because they lack, or only have one, chlorine inortho-positions. A toxic equivalency (TEQ) value could be calculated for a mixture of
PCDDs, PCDFs, and dioxin-like PCBs, assuming that their effects are additive [7,8]. Hence,
the TEQ value is the sum of the TEF value for each congener multiplied by its
concentration. Use of TEQ values facilitates risk assessments and the drafting andimplementation of legislation regarding mixtures of dioxin-like compounds.

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Several in vitro bioassays have been developed for assessing AhR activity and the detection
and quantification of dioxins and related chemicals [9–12]. The primary advantages of AhRbased bioassays are their high throughput capacity and low cost, compared with gas
chromatography/high-resolution mass spectrometry (GC/HRMS) [12]. However, deviations
between the TEF values for individual congeners and their relative effective potency to
induce a bioassay response, and contributions from AhR ligands for which no TEFs areavailable, could in principle lead to discrepancies between bioassay and GC/HRMS results.
Nevertheless, these bioassays have been successfully used in several applications, including
assessments of dioxin-like potency in sediments and pore water, blood plasma, sewage
sludge, food control, and analyses of polycyclic aromatic hydrocarbons (PAHs) [13–17].
Intra- and interlaboratory comparison studies have confirmed their usefulness in dioxin
analysis of soils and sediments [18–20]. Moreover, the performance of a CYP1A1-dependent
ethoxy-resorufin O-deethylase (EROD) bioassay and an immunoassay for TEQ
determination of environmental samples, including contaminated soil, has also been
compared [21,22].
The chemically activated luciferase expression (CALUX) and chemically activatedfluorescence expression (CAFLUX) cell bioassays are two state-of-the-art techniques thatcan be used to generate bioassay TEQ values (CA[F]LUX-TEQs) that can be compared with
GC/HRMS TEQ estimates [23–26]. Both of these bioassays involve the use of geneticallymodified mouse hepatoma cells, and after exposure to sample extracts containing AhR
ligands, they produce luciferase or enhanced green fluorescent protein, respectively. The
intensity of the luminescence or fluorescence signal is directly proportional to the amount of
AhR ligands in the sample and can be converted to a CA(F)LUX-TEQ by comparison to a
TCDD calibration (standard) curve. The critical parameters that influence the CALUX
results have been described elsewhere [27]. For environmental samples, sample preparation
steps including extraction, clean-up, and fractionation have a significant effect on thecomposition of AhR ligands in the final extract, constituting a complex mixture ofEnviron Toxicol Chem. Author manuscript; available in PMC 2010 April 23.

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compounds. Because many compounds produce AhR-independent toxic and biologicalresponses and many AhR-active chemicals have not been assigned TEFs [4–6], it isimportant to purify extracts to eliminate such interfering substances to avoid potential bias indata interpretation.
In the study presented here, we evaluated the utility of three different bioanalyticaltechniques for dioxin analysis of aged, contaminated soil samples (an enzyme-linked
immunosorbent assay [ELISA] [28] and two cell-based bioassays [CALUX and CAFLUX])
by comparing results obtained from these methods with PCDD/F data obtained by GC/
HRMS analysis (the PCBs were only present in a limited number of samples and therefore
not included in the comparison). To facilitate rigorous comparisons, identical conventional
GC/HRMS preparation protocols were applied to all of the samples analyzed by all of theexamined methods. This procedure includes Soxhlet extraction followed by multistep
column clean-up with American and European standard methods [29,30]. Several fractionsin addition to those containing PCDD/Fs were collected and tested to screen for the presenceof AhR agonists and antagonists. To the best of our knowledge, this is the first time thatthese PCDD/F bioanalytical techniques have been tested in parallel with identical purified
extracts. The overall focus of this work was on evaluating the quantitative performance of
the ELISA and the CALUX and CAFLUX bioassays and to examine the possible effect ofsample purification and data interpretation on the results.

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MATERIALS AND METHODS
Chemicals

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Solvents used for extraction and clean-up (toluene, n-hexane, dichloromethane, methanol)
were of glass-distilled grade and supplied by Honeywell Burdick and Jackson (Muskegon,
MI, USA). Tetradecane (olefin-free, p.a. grade) was from Fluka (Buchs, Switzerland), anddimethyl sulfoxide (DMSO, analytical grade) was from Scharlau (Barcelona, Spain). Silica(Kieselgel 60) and anhydrous sodium sulfate were from Merck (Darmstadt, Germany). The
AX-21 carbon originated from Anderson Development (Adrian, MI, USA) but is currently
not commercially available. For the cell bioassays, tissue culture media and geneticin(G418) were supplied by Invitrogen/Gibco (Carlsbad, CA, USA), fetal bovine serum (FBS)
by Atlanta Biologicals (Atlanta, GA, USA), and luciferase lysis buffer and luciferin reagent
by Promega Corporation (Madison, WI, USA). The TCDD that was used in the CALUX
assay was a gift from S. Safe (Texas A&M University, TX, USA). The TCDD used in the
CAFLUX assay and isotopically labeled standards used for GC/HRMS analyses were from
Cambridge Isotope Laboratories (Andover, MA, USA). For the GC/HRMS quantification oftarget analytes, a standard solution containing the 17 native 2,3,7,8-substituted PCDD/Fs
was used (Wellington Laboratories, Guelph, ON, Canada).
Samples
Soil samples were collected from five wood treatment sites (wood 1–10) and from a chloralkali site (chlor 1 and 2) in Sweden. All wood treatment sites were located in northern
Sweden and were polluted with pentachlorophenol (PCP), 2,3,4,6-tetrachlorophenol (TCP),
or both wood impregnation formulations containing considerable amounts of dioxins,
especially hepta- and octa-CDD/Fs. A sample of an artificial soil containing 10% peat, 20%
kaolin, and 70% sand with background levels of PCDD/Fs prepared by Pelagia Miljökonsult(Hörnefors, Sweden) according to guidelines published by the Organisation for Economic
Co-operation and Development (OECD) was also included. All samples were air-dried atroom temperature, passed through a 2-mm sieve, and thoroughly homogenized before
extraction. The dry weight of the samples was determined by weighing portions of them
before and after heating for at least 12 h at 105°C.

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Soxhlet extraction and clean-up

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Air-dried samples (2.5–5 g) were Soxhlet-extracted with toluene for 15 h, and each extract
was split into aliquots for analysis by GC/HRMS, the two cell-based bioassays, and an antidioxin antibody-based ELISA. The sample clean-up procedure preceding all of the different
detection techniques was identical, except for the addition of internal standards to the
portions intended for GC/HRMS. The purified portions without internal standards were split
into three separate aliquots for analysis by CALUX, CAFLUX, and ELISA. One of theoriginal crude extract aliquots was not subjected to sample cleanup and purification and was
directly examined by the CAFLUX assay for determination of total AhR agonist activity.

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The clean-up procedure has previously been described in detail [31]. Briefly, the bulk of thecoextracted material was removed by a multilayer column sequentially packed in-house with
35% KOH-silica (w/w), activated silica, and 40% sulfuric acid–silica (w/w) then eluted withn-hexane. The solvent was evaporated to about 1 ml and applied to a carbon column(AX-21/Celite, 1:12, w/w), which was eluted with dichloromethane/n-hexane (1:1, v/v). The
column was then turned upside down and eluted with toluene. The toluene eluate was
evaporated to about 1 ml and applied to an alumina oxide column, which was eluted with nhexane and dichloromethane/n-hexane (1:1, v/v). As a final step, the dichloromethane/nhexane fraction was evaporated to 0.5 ml; applied to a miniaturized multilayer column;
sequentially packed with 35% KOH-silica (w/w), activated silica, and 40% sulfuric acidsilica (w/w); and eluted with n-hexane. The solvent of spiked aliquots of the extracts waschanged to tetradecane (50 µl), and the solvent of nonspiked aliquots to DMSO (30 to 800µl). The losses during solvent exchange were minimized by avoiding evaporation to dryness.
Earlier evaluations have shown that the analyte losses are <10% with such an approach.
GC/HRMSThe instrumental analysis by GC/HRMS was carried out as previously described [31].
Selected ion monitoring at a resolution of 8,000 to 10,000 was used to quantify the 2,3,7,8substituted PCDD/F congeners by the isotope dilution technique. The target analytes wereidentified by checking retention times and isotope ratios.
ELISA

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The protocol of an ELISA analysis with the use of 2,3,7-trichloro-8-methyl-dibenzo-pdioxin (TMDD) as surrogate standard has been described elsewhere [28]. The ELISA
analysis was performed at the University of California (Davis, CA, USA). Triplicates ofserial dilutions of sample extracts and standards were analyzed. The progressive inhibitionof the assay caused by increasing the amounts of soil in the tested extract was related to the
TMDD standard concentration–absorbance curve, and the results were converted to TMDD
equivalents and interpreted as total PCDD/F contents. Calculated mean values and standard
deviations of PCDD/F contents in the soil samples were all based on individual absorbancemeasurements of each tested dilution.
CALUX
Genetically modified mouse hepatoma cells (H1L6.1c3) were used in the CALUX cellbioanalysis [26,32]. These cells contain a stably transfected AhR-responsive firefly
luciferase gene that responds to TCDD and other AhR agonists by expressing luciferase in atime-, dose-, and chemical-specific manner. The CALUX analysis was performed at
University of California, Davis. Briefly, a portion (100 µl) of cell suspension containing
75,000 cells was added to each well of a Costar white, 96-well, clear-bottomed microplate(Corning, NY, USA) and incubated at 37°C for 24 h before sample addition. Serial dilutionsof sample extracts and a standard (TCDD in DMSO, 0.5 to 5,000 pM) in FBS-supplemented

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cell media were then added to the wells of the plate. All dilutions were tested in triplicate.
The final concentration of DMSO was 1% (v/v). Cells were incubated at 37°C withtreatment media for 24 h, then lysed with Promega luciferase lysis buffer, and the luciferase
activity was initiated by the automatic injection of Promega stabilized luciferase reagent.
The light output was then measured with an automated microplate luminometer (Anthos
Lucy2, Eugendorf, Austria) and expressed as relative light units.
CAFLUXThe CAFLUX analysis was performed at the Swedish Defense Research Agency (Umeå,Sweden) with the use of genetically modified mouse hepatoma cells (H1G1.1c3) according
to protocols described elsewhere [24,25]. Briefly, 70,000 cells were transferred to each wellof a Nunc black, 96-well, clear-bottomed microplate (Nunc, Roskilde, Denmark) andallowed to attach for 24 h before exposure to TCDD or soil sample extracts. The cells werethen treated with serial dilutions of the sample extracts or TCDD in DMSO without
removing the cell culture media. A TCDD standard curve (0.3–2,000 pM) was created foreach plate, and all dilutions were tested in triplicate, with a final concentration of 1% (v/v)
DMSO. The background fluorescence in the intact cells was determined immediately after
dosing and after incubation for 24 h at 33°C. The fluorescence in each well of the plate wasmeasured in a microplate fluorometer (Fluostar Galaxy, BMG Labtechnologies, Offenburg,
Germany) with an excitation wavelength of 485 nm and an emission wavelength of 520 nm.

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CA(F)LUX-TEQ calculations

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The CA(F)LUX-TEQ values for effective concentrations (ECs) were calculated at 25, 50,
and 75% (EC25, EC50, and EC75) of the maximum responses on the TCDD dose–response
curve. To obtain the mean CA(F)LUX-TEQ values and standard deviations for the samples(pg/g sample), the EC25, EC50, and EC75 values from the TCDD curves were divided by
the amount of soil corresponding to the EC25, EC50, and EC75 response levels of thesample dose–response curves, after which the mean and standard deviations of these
quotients were calculated. Both the TCDD standard curves and the sample curves werefitted with the use of SigmaPlot 2001 version 7.101 software (Systat Software, Richmond,
CA, USA), to the four-parameter Hill function. For two of the CAFLUX-analyzed samples(wood 5 and wood 6), the EC75 levels were extrapolated. Furthermore, to obtain complete
sample dose–response curves for four of the CAFLUX-analyzed samples (wood 2, 3, 9, andchlor 2), the response at the background level (derived from DMSO-treated cells) was usedin the curve fitting (because even the most diluted extract produced a signal considerably
higher than background). For samples that did not produce a complete dose–response curve,
the CA(F)LUX-TEQs were calculated with the use of the individual responses of all
dilutions that produced a response in the EC25 to EC75 range.
In a few cases, the inducing activity was very low, so the CA(F)LUX-TEQ values were
calculated with responses below the EC25 level. However, even in these cases, the response
was higher than the limit of quantification, which was defined as the mean response of
triplicate determinations of the DMSO control plus 10 times the standard deviation.

RESULTS AND DISCUSSION
Comparison of the bioassay, immunoassay, and gas chromatography/mass spectrometrydata
A standard (TCDD or TMDD) and a sample extract curve (wood 1) of the CALUX and
CAFLUX assays and the ELISA are presented in Figure 1. The TCDD used in the CALUX
analysis (in USA) and CAFLUX analysis (in Sweden) was of different origin but did give
similar responses. In some cases, the concentration of PCDD/Fs in the sample extract was
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not enough to generate complete assay curves. Although this could have been compensatedfor by the use of larger sample intakes, this was not done for practical reasons because
complementary tests were difficult to perform because the tests had been done in anothercountry (ELISA and CALUX).
The results of the ELISA, CALUX, and CAFLUX, together with GC/HRMS reference
values, for aged dioxin–contaminated soil samples are compiled in Figure 2. The reported
CA(F)LUX-TEQs represent the mean values calculated from those determined from several
EC values (i.e., EC25, EC50, EC75) derived by fitting the standard and sample extract
curves to the four-parameter Hill function, with the following exceptions: CALUX results
for five of the samples (woods 6–10) and CAFLUX results for four samples (woods 7, 8, 10,
and artificial soil), for which PCDD/F calculations were based on single-point estimatesfrom the respective standard curves. The error bars correspond to the standard deviations for
multiple CA(F)LUX and ELISA determinations of serial dilutions of sample extracts, except
for the CALUX determination of wood 1 (n = 2) and the CAFLUX determinations of wood
7 (n = 2), for which the range of the two individual values are indicated. The luminescencefor wood 5 and the artificial soil were below the detection limit. Also the inhibition of theabsorbance for woods 4 to 10 and the artificial soil were below the detection limit.

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Because ELISA failed to detect PCDD/Fs in eight of the 13 analyzed samples, it is clearly
the least sensitive assay. However, in several cases, the ELISA response was very close to
the detection limit, suggesting that an increased sample intake would have allowed ELISA
detection. For two of the tested samples (wood 5 and the artificial soil), CALUX also failed
to give a detectable response. Thus, CAFLUX appeared to be the most sensitive of thebioanalytical techniques.
Statistical evaluation of all data (excluding nondetects) by t test revealed no significant
differences between the methods (p = 0.05), except for GC/HRMS and ELISA (p = 0.02).
The correlation coefficients (r2) were also calculated by linear regression for all possiblecombinations of detection methods (Table 1). The analyses show that CALUX was mostcorrelated with GC/HRMS, followed by ELISA and CAFLUX. Furthermore, all the data of
the bioanalytical techniques were highly correlated (r2 > 0.92). It has to be stressed,
however, that the ELISA underestimated all data by an order of magnitude and that thecorrelation analysis might be compromised because of the limited ELISA data set.

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The data suggest that CALUX and CAFLUX produce sufficiently accurate data to meetcriteria set by the U.S. Environmental Protection Agency for screening samples containing
higher levels of dioxins than the remediation threshold of 1,000 pg PCDD/F TEQs/g forresidential soils [33]. The ELISA underestimation of the dioxin content by roughly an orderof magnitude (see above) does, however, call for the use of site-specific correction factors.
Previous investigations of the studied soils, together with other contaminated soils, haveshown that ELISA can also provide acceptable results if the data are corrected for
differences between the TEF and cross-reactivity values of the PCDD/Fs present in thesamples [28]. Hence, the two bioassays, and possibly the ELISA, could be used forscreening PCDD/Fs in contaminated soil samples and for prioritizing samples for further
analysis by GC/HRMS, thereby increasing the overall number of samples that can bescreened in large-scale soil pollution surveys. For such activities, the ELISA approach
would have considerable advantages because it could be most easily adapted for use in thefield, which is a highly desirable feature. However, the bioassays provide more accurate
determinations of relatively lightly contaminated soils, and biologically relevant sums of
AhR ligands are reported. In addition, the CAFLUX bioassay is significantly cheaper andmore convenient than CALUX and yields realtime induction responses. Drawbacks of thecell-based bioassays include restrictions on their application because of their use ofEnviron Toxicol Chem. Author manuscript; available in PMC 2010 April 23.

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genetically modified cells and the limited amounts of validation data that are currently
available, especially for the CAFLUX assay. Furthermore, the CAFLUX assay produceshigh background fluorescence, which might influence the results of lightly contaminatedsamples.
Bioassay data evaluation
The sample and TCDD dose–response curves yielded by the bioassays were not always
parallel, and the efficacy (maximal induction response) indicated by the curves was not
identical, in accordance with previous reports [34]. The discrepancies are illustrated by thedifferences in the slopes and maximal induction responses between the soil dose–response
curve for wood 1 and the TCDD dose–response curve shown in Figure 1a and b.
Consequently, the CA(F)LUX-TEQ value was dependent on the dilution chosen for
quantification; thus, the relative potency at the EC25-, EC50-, and EC75-based values
differed somewhat. In Figure 3a, the CAFLUX-TEQs calculated with the use of EC25,
EC50, and EC75 values for all samples that gave complete or nearly complete dose–
response curves are shown, and the corresponding data from the CALUX assay are shown in
Figure 3b. Parallel standard and sample curves prevail when calculated values are very
close. In contrast, if the values are widely dispersed, the curves are nonparallel, whichsuggests nondioxin compounds that influence the assay response. Thus, such samples might
benefit from improved clean-up.

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The effect of cleaning sample extracts on the bioassay results

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The AhR can be activated by a structurally diverse range of chemicals, of which the PCDD/
Fs are only one class [4–6,35]. Accordingly, accurate detection of PCDD/Fs in sample
extracts requires clean-up of the extracts to remove the undesired “nonclassical” AhR
ligands, which often are moderate to weak AhR inducers compared with TCDD [4–6].
These might otherwise contribute significantly to the overall bioassay induction, leading tooverestimation of the potency of the extract if they are not removed. As expected, thesample cleanup procedure applied in this study removed large quantities of such interferingor undesired compounds, as illustrated by the significantly weaker (sometimes up to 100fold weaker) CAFLUX responses induced by purified extracts than by crude extracts (Table2). Hence, the overall activity of the crude extracts results from both classical andnonclassical AhR ligands [35] present in the extract, and accurate analysis of the target
PCDD/Fs required removal of the undesired AhR agonists in the multistep column clean-up
process. Although the CA(F)LUX-TEQs derived from the cleaned-up extracts should result
primarily from the toxicologically relevant PCDD/Fs, and thus be comparable to the GC/
HRMS-TEQ of the 2,3,7,8-PCDD/Fs, the possibility of contributions from other dioxin-like
AhR agonists (i.e., polychlorinated naphthalenes) that could copurify with the PCDD/Fs
could not be excluded. However, in this study, the contributions of other halogenatedaromatic hydrocarbons to the overall response seemed to be limited because there was good
agreement between CA(F)LUX and GC/HRMS data. Nevertheless, unwanted substances in
the purified extracts could still be the reason for nonparallel curves, impairing accuratequantification. An improved cleanup on the basis of new protocols, or on a refinement of thepresent, would therefore be beneficial and might lead to improved CAFLUX performancefor samples currently being severely overestimated by this method, especially samples
containing the lowest amounts of PCDD/Fs (e.g., woods 9 and 10).
The CAFLUX responses to other fractions produced during the clean-up were alsomeasured. The first fraction eluted from the carbon column, which would be expected tocontain any multi–ortho-substituted PCBs and polychlorinated diphenylethers (PCDEs)present in the sample, did not induce any significant CAFLUX response, although GC/
HRMS analysis of three of the samples revealed significant amounts of PCDEs (270–1,100

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ng/g dry wt, not shown). Thus, the CAFLUX assay did not recognize PCDEs as AhR
agonists and this is consistent with findings reported by other authors [36]. Furthermore, thedetection of the non–ortho-PCB 3,3′,4,4′-tetrachlorobiphenyl (PCB77) in the dioxin fractionof the chlor-alkali site samples (at high picogram levels) demonstrated that AhR-active
PCBs were also present in these samples. Because the multi–ortho-PCBs should elute in thefirst carbon column fraction, together with the PCDEs, these compounds would notcontribute to the overall CAFLUX response to the cleaned-up sample extracts.
Tests for antagonism and other potential biasing factors
Because antagonists can suppress the AhR-dependent induction response [37], CAFLUX
cells were exposed to DMSO or sample extracts (first fraction eluted from the carbon
column, n = 4) spiked with known amounts of TCDD to evaluate whether the observed zero
response was due to antagonists. In all cases, the magnitude of the response was correlatedwith the amount of TCDD added, indicating that inhibitory chemicals were not present in
the extracts (data not shown).

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In CAFLUX analysis, the signal might also be biased because of the presence of fluorescent
substances, compounds that quench fluorescence, or both. However, no fluorescence was
detected in any wells immediately after addition of the sample extracts, indicating that no
fluorescent chemicals were present in the soil extracts that could affect accurate
measurements of enhanced green fluorescent protein. Furthermore, no reduction in the
fluorescence signal produced in TCDD-treated cells during the course of the 24-h
incubations was detected after addition of the sample extracts, indicating that no
fluorescence-quenching compounds were present in the crude sample extracts.

CONCLUSIONS

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The ELISA and CA(F)LUX assays are bioanalytical techniques that could be used in
combination with instrumental analysis to identify and characterize environmental andbiological samples that contain PCDD/Fs. Given the ability of these techniques to also
recognize other compounds, they will not be able to replace instrumental analysis methods.
However, they can greatly facilitate monitoring studies when they are used to prescreen
sample extracts to identify potentially contaminated samples and to prioritize samples for
subsequent congener-specific instrumental analysis by GC/HRMS. Consequently, thesebioanalytical techniques might be very useful in remediation programs for PCDD/F-polluted
industrial areas when rapid screening of large numbers of samples is essential. It is worth
noting that the bioassays will respond to other dioxin-like AhR agonists if present in thesamples, thereby facilitating the discovery and identification (by GC/HRMS) of previously
unknown compounds (in the dioxin fraction) with dioxin-like biological effects. The dioxinlike PCBs, especially the non–ortho-PCBs coeluting with the PCDD/Fs in the current cleanup, will, for instance, cause CA(F)LUX response if present in the samples. This might be thereason for the over-estimation of PCDD/F content in the chlor-alkali samples because thesecontain significant amounts of non–ortho-PCBs. However, these compounds are not present
to any significant extent in the rest of the samples and consequently do not interfere with theCA(F)LUX-TEQ determinations of the wood treatment samples.
To maximize the benefits of the rapid bioanalytical techniques they should be combinedwith high-throughput extraction and clean-up techniques. We have therefore developed asimultaneous extraction and fractionation technique for PCDD/Fs in contaminated soils withthe use of selective accelerated solvent extraction (ASE), and it has been successfully
combined with the ELISA detection method [28]. A similar selective ASE method has beendeveloped and used in combination with CAFLUX for determining dioxin TEQs in food andfeed samples [38]. Currently, the performance of selective ASE of soil samples withEnviron Toxicol Chem. Author manuscript; available in PMC 2010 April 23.

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Page 9CAFLUX detection is being evaluated, and a significant reduction in costs and increase inthroughput is expected.

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Finally, it has to be stressed that the CA(F)LUX-TEQ levels that have been obtained in thisstudy relate to the total amount of bioactivity present in the soils, and this does notnecessarily reflect the toxicity, but merely gives information on the potential for geneinduction. Although AhR-dependent gene induction is a prerequisite for AhR-dependent
toxicity, additional analysis by instrumental, in vivo animal studies, or both must be carriedout before any definitive conclusions regarding the toxicity of a sample or extract can be
made. Accordingly, accurate exposure and risk assessment analysis would also require
information on the bioavailability of the target pollutant(s), which might be obtained throughthe use, for instance, of mild extraction techniques.

Acknowledgments
The Swedish Research Council for Environment, Agricultural Sciences, and Spatial Planning (FORMAS), The
Swedish Association of Graduate Engineers (CF), and the U.S. National Institutes of Environmental Health
Sciences (ES07685 and ES04699) are gratefully acknowledged for financial support. This work was carried out
within the framework of the North Sweden Soil Remediation Centre (MCN).

REFERENCES
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Fig. 1.

(a) Chemically activated luciferase expression (CALUX), (b) chemically activatedfluorescence expression (CAFLUX), and (c) enzyme-linked immunosorbent assay (ELISA)
dose–response curves for the standard 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) or 2,3,7trichloro-8-methyl-dibenzo-p-dioxin (TMDD) and the wood 1 soil extract, illustrating theslight deviations from parallelism between the CA(F)LUX standard and sample extract
curves and an incomplete ELISA sample extract curve. All data have been corrected forsignals from a blank sample (wells treated with dimethyl sulfoxide).

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Fig. 2.

Comparisons of results of gas chromatography/high-resolution mass spectrometry (GC/HRMS) World Health Organization toxic equivalency (WHO-TEQ) reference values (pg/g
dry wt) and chemically activated luciferase expression (CALUX)–TEQ (pg/g dry wt),chemically activated fluorescence expression (CAFLUX)–TEQ (pg/g dry wt), and enzymelinked immunosorbent assay (ELISA) analysis of 2,3,7-trichloro-8-methyl-dibenzo-p-dioxin(TMDD) equivalents (pg/g dry wt). The GC/HRMS results were calculated with TEQ
factors from Van den Berg et al. [7], including only dioxins. For comparison purposes, the±30% uncertainty in the accredited GC/HRMS procedure used was included in the graph.

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Fig. 3.

Bioassay toxic equivalency (TEQ) concentration ranges for soil samples analyzed by (a)chemically activated fluorescence expression (CAFLUX) and (b) chemically activatedluciferase expression (CALUX). The uncertainties in the determinations because ofdeviations from parallelism between the sample and standard curves are indicated by thesize of the range. Data points outside the bars were derived by extrapolation beyond theempirical data. W = wood; C = chlor.

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Table 1

Correlation coefficients for the methods under study

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GC/HRMSaCALUXb

GC/HRMSCALUXCAFLUX

ELISA

1

0.719

0.617

0.655

1

0.923

0.927

1

0.999

CAFLUXc
ELISAd1a

Gas chromatography/high-resolution mass spectrometry.

b

Chemically activated luciferase expression.

c
Chemically activated fluorescence expression.
dEnzyme-linked immunosorbent assay.

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Table 2

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Results of chemically activated fluorescence expression (CAFLUX) analysis of soil samples from wood
treatment sites and a chlor-alkali site in Sweden expressed as CAFLUX toxic equivalency values (TEQs)
CAFLUX TEQs (pg/g dry wt)
Sampling site
Wood

Chlor

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Artificial soil

Crude extract

Dioxin fractiona

1210,000

12,0002

100,000

33,0003

230,000

8,200

41,600b

80

5

710

10

6

6,800

76

7

12,0001,000

860,0001,000

91,600b1,000

1012,0001,100

144,0003,300

21,000,000b

490,000

2,400b12a

For details regarding the quantification, see text.

b

On the basis of individual dilutions of samples (n = 2–4).

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