NIH Public Access
Author Manuscript
Talanta. Author manuscript; available in PMC 2012 February 15.

NIH-PA Author Manuscript

Published in final edited form as:
Talanta. 2011 February 15; 83(5): 1415–1421. doi:10.1016/j.talanta.2010.11.035.

Considerations for potency equivalent calculations in the Ah
receptor-based CALUX bioassay: Normalization ofsuperinduction results for improved sample potency estimation
David S. Baston and Michael S. Denison*
Department of Environmental Toxicology, University of California, Davis, CA 95616, USA

Abstract

NIH-PA Author Manuscript

The chemically activated luciferase expression (CALUX) system is a mechanistically based
recombinant luciferase reporter gene cell bioassay used in combination with chemical extraction
and clean-up methods for the detection and relative quantitation of 2,3,7,8-tetrachlorodibenzo-pdioxin and related dioxin-like halogenated aromatic hydrocarbons in a wide variety of sample
matrices. While sample extracts containing complex mixtures of chemicals can produce a variety
of distinct concentration-dependent luciferase induction responses in CALUX cells, these effects
are produced through a common mechanism of action (i.e. the Ah receptor (AhR)) allowing
normalization of results and sample potency determination. Here we describe the diversity in
CALUX response to PCDD/Fs from sediment and soil extracts and not only report the occurrenceof superinduction of the CALUX bioassay, but we describe a mechanistically based approach fornormalization of superinduction data that results in a more accurate estimation of the relative
potency of such sample extracts.

Keywords
2, 3, 7, 8-Tetrachlorodibenzo-p-dioxin; TCDD; Ah receptor; CALUX; Bioassay; Superinduction

1. Introduction
NIH-PA Author Manuscript

Halogenated aromatic hydrocarbons (HAHs), such as the poly-chlorinated dibenzo-pdioxins (PCDDs), dibenzofurans (PCDFs) and biphenyls (PCBs), are a environmentally andmetabolically persistent class of highly toxic chemicals. Exposure to 2,3,7,8tetrachlorodibenzo-p-dioxin (TCDD, dioxin), the prototypical and most potent HAH, andrelated “dioxin-like” HAHs (dl-HAHs) can produce a wide variety of species and tissuespecific toxic and biological effects, including immuno- and hepato-toxicity, endocrine
disruption, cancer and lethality [1–5]. It has been well established that the majority of the
toxic/biological effects of dl-HAHs are mediated by the Ah receptor (AhR), a soluble
intracellular ligand-dependent transcription factor [1,2,5–8], and that AhR-dependent
toxicity requires persistent activation of the AhR signaling pathway by metabolically stable
dl-HAHs [1,2,5,9].
When assessing the toxicity and analysis of HAHs present in environmental and biologicalsamples, one must take into consideration that there exists a total of 209 different PCB, 135different PCDF and 75 different PCDD congeners, of which TCDD-like biological/
toxicological effects are produced by a relatively small number of these congeners [2,10].
*Corresponding author at: Department of Environmental Toxicology, Meyer Hall, One Shields Avenue, University of California,
Davis, CA 95616-8588, USA. Tel.: +1 530 752 3879; fax: +1 530 7523394. msdenison@ucdavis.edu (M.S. Denison).

Baston and Denison

Page 2

NIH-PA Author Manuscript

Toxic equivalency factors (TEFs) for those individual HAH congeners producing in vivo
“dioxin-like” toxicity have been established by the World Health Organization and thesevalues represent the toxic potency of the specific dl-HAH congener expressed relative to thatof the most toxic congener, TCDD [10]. Determination of the toxic potency of dl-HAHspresent in a given sample extract first requires the application of sophisticated cleanup
procedures followed by high-resolution instrumental analysis (capillary gas chromatography
and high resolution mass spectrometry (GC/HRMS)) for the separation, identification, andquantitation of individual PCDD, PCDF and PCB congeners [11–14]. Calculation of therelative toxic equivalency (TEQ) or toxic potency of a complex mixture of dl-HAHs has
been extensively described and reviewed [2–10,14] Although the instrumental analysis
methodology approach is considered the “gold standard” for measurement of HAHs insample extracts and ultimately for determination of sample TEQs, these analyses require
highly sophisticated equipment and training and can be very costly and time-consuming.
Accordingly, numerous rapid and relatively inexpensive in vitro and cell-based bioanalytical
approaches have been developed that are capable of detecting and estimating the relative
potency of complex mixtures of dl-HAHs, many of which take advantage of the AhRdependent signal transduction mechanism of action [13–20].

NIH-PA Author Manuscript
NIH-PA Author Manuscript

The chemically activated luciferase expression (CALUX) recombinant cell bioassay is one
such bioassay system that has been extensively utilized for the detection and relative
quantitation of dl-HAHs in sample extracts, although numerous other cell bioassays havebeen described (reviewed in [13,14,17–19,21]). The CALUX bioassay system takes
advantage of the AhR-dependent signal transduction mechanism of action of TCDD and dlHAHs and utilizes recombinant mammalian cell lines that contain a stably transfected AhRresponsive firefly luciferase reporter gene that responds to dl-HAHs and other known AhR
agonists with the induction of luciferase in a dose-, time-, chemical- and AhR-dependent
manner [13,18,19,21]. Although the highest affinity and most potent activators of the AhR
signaling pathway are TCDD and related dl-HAHs, recent evidence has shown that the AhR
can be activated by an extremely diverse range of chemical ligands whose structure andphysiochemical characteristics are dramatically different than that of dl-HAHs [8,20,22–24].
Accordingly, accurate assessment of the relative potency of dl-HAHs present in a complex
chemical mixture requires removal of undesired AhR active substances by chemical
fractionation and clean-up procedures [13,14,19,25–27]. The CALUX bioassay whencoupled with an appropriate sample extract clean-up method has been used successfully for
accurate detection and relative quantitation of dl-HAHs in a wide variety of biological,
environmental and food/feed samples (reviewed in [13,19]) and has received regulatory
certification as a validated method by the US EPA (Method 4435) for determination of dlHAHs in various matrices [28]. Numerous studies have reported that potency estimates for agiven sample determined by CALUX and GC/HRMS typically exhibit a reasonably highdegree of correlation [13,19,26,27]. However, these same studies also revealed that potency
estimates determined by CALUX and other cell bioassays commonly yield values (referredto here as bioanalytical equivalents (BEQs)) significantly higher than the TEQs calculated
from GC/HRMS analysis of the same samples, particularly when environmental samples areanalyzed. This overestimation by the bioassays has been suggested to result from differences
between the TEF values for the dl-HAHs used for GC/HRMS TEQ calculations and theirCALUX-based relative potency (REP) values [29] and/or the presence of other AhR active
dl-HAHs (i.e. polybrominated and/or mixed chloro/bromo-dl-HAHs or other chemicals[19,30–32]) in sample extracts that are not measured using GC/HRMS methods establishedfor chlorinated dl-HAHs. The recent demonstration that activation of protein kinase C andinhibition of protein synthesis can synergistically enhance AhR-dependent gene expression[33–37] suggests additional avenues by which chemicals in a complex mixture can enhancethe output response of the CALUX bioassay leading to an inaccurate overestimation ofsample potency (i.e. BEQ). While synergistic enhancement of AhR-dependent gene
Talanta. Author manuscript; available in PMC 2012 February 15.

Baston and Denison

Page 3

NIH-PA Author Manuscript

expression can be produced using purified compounds and these mechanisms in controlled
experimental conditions, their relevance and significance for CALUX bioassay analysis ofactual environmental samples remains to be confirmed. Here we report for the first timesuperinduction of the CALUX bioassay by the PCDD/Fs fraction extracted from soil/
sediment samples and describe a mechanistically based approach for normalization ofsuperinduction data that results in a more accurate estimation of the relative potency (BEQ)of such sample extracts.

2. Methods and materials
2.1. Chemicals and materials
TCDD was a kind gift from Dr. Stephen Safe (Texas A&M University). Luciferase substrate(luciferin) and lysis buffer were purchased from Promega, alpha minimum essential media(αMEM) from Gibco/Invitrogen and fetal bovine serum (FBS) from Atlanta Biologicals.
White Costar 96 well clear-bottomed tissue culture plates, pesticide residue grade solvents,
silica gel, celite and granular sodium sulfate were purchased from Fisher Scientific. Sodium
sulfate was baked at 450 °C for 4 h in an aluminum tray prior to use. All experimental
glassware was borosilicate and was prepared by hand washing with a final triple rinse ofMilli-Q water, air drying, and openings and ground glass contact surfaces covered withheavy duty aluminum foil and baked in a muffle furnace for a minimum of 4 h at 450 °C.

NIH-PA Author Manuscript

2.2. Sediment/soil sample extract preparation

NIH-PA Author Manuscript

Sediment and soil samples from a previous study [38] were obtained in a crushed, dry state
ready for extraction. Approximately 2 g of sample was extracted with 10 ml of 80:20
toluene:methanol, followed by two extractions with 10 ml of toluene each. Individual
extracts were allowed to settle for 20–30 min followed by decanting of the supernatant onto
a layered filter column consisting of (from the bottom of the column up): glass wool fiber,baked sodium sulfate, celite and baked sodium sulfate (for removal of particulate material).
The column was rinsed with an additional 10 ml of toluene and all flow-through eluateswere combined and the solvent volume reduced to near dryness by vacuum centrifugation.
Sample extracts were rinsed from the centrifuge tube and resuspended with 2 ml of hexane
followed by four more 1 ml hexane rinses. Each rinse was transferred onto a 33% acid silica
column where all rinses were composited, allowing for the separation of AhR active
polycyclic aromatic hydrocarbons [27,28] from HAHs. The acid silica column was directly
eluted onto an XCarb column (celite/1% XCarb column sandwiched between sodium sulfate
and retained between glass wool plugs [27,28]), followed by three 5 ml hexane rinses of theacid silica column. This column combination allows for the separation of dl-HAHs into
PCDD/F and PCB fractions [28]. The XCarb column was then separately rinsed with anadditional 5 ml hexane solvent aliquot. Two separate fractions were eluted from the XCarb
column: first PCBs and PCB-like compounds were eluted from the XCarb column with 15
ml of a 80/10/10 hexane/toluene/ethyl acetate solution, the XCarb column was then inverted
and PCDD/Fs eluted from the XCarb column with 15 ml of toluene. All solvents werereduced to near dryness by vacuum centrifuge. Samples were rinsed from the centrifuge tube
with 1 ml of hexane and this was repeated 3 more times (a total of 4 ml), each 1 ml rinsetransferred to an amber, Teflon-capped 4 ml vial, sealed with parafilm, and stored wrappedin foil in the dark until analyzed. Only the PCDD/F-containing fraction was used for the
CALUX bioassay studies described here. The concentration of PCDDs and PCDFs in thesesamples had been previously determined using standard GC/HRMS analysis [38] and TEQ
values for each sample calculated using the 2005 TEF values [10].

Talanta. Author manuscript; available in PMC 2012 February 15.

Baston and Denison

Page 42.3. Cell culture, chemical treatment and CALUX analysis

NIH-PA Author Manuscript
NIH-PA Author Manuscript

Recombinant mouse hepatoma (H1L6.1c3) CALUX cells were grown and maintained as wehave previously described [18,21,28]. These cells contain a stably integrated dioxin
responsive element (DRE)-driven firefly luciferase reporter plasmid, pGudLuc6.1, that
responds with the induction of luciferase gene expression in a ligand-, dose- and AhRdependent manner [18,19,21]. Cells were plated into white, clear-bottomed 96-well tissue
culture dishes at 75,000 cells/well and allowed to attach for 24 h, then incubated in triplicate
with carrier solvent DMSO (1% final concentration), the indicated concentration of TCDD
or the indicated mass equivalent of sediment/soil extract (the PCDD/F fraction) for 24 h at
37 °C. For luciferase measurement, treatment media was removed from the sample wells,
the wells rinsed with 100 μl phosphate-buffered saline followed by addition of 50 μl of
Promega cell lysis buffer and mixing on an orbital shaker for 20 min at room temperature toensure complete cell lysis. Treated 96-well plates were either analyzed immediately or
stored frozen at −80 °C. Measurement of luciferase activity in each well (integrated over 10
s after a 10 s delay) was carried out using either a Berthold or Anthos Lucy2 microplate
luminometer with automatic injection of 50 μl Promega stabilized luciferase reagent.
Luciferase activity in each well was expressed relative to that maximally induced by 1 nM
TCDD after background correction. All plates contained a standard curve of at least nineconcentrations, in triplicate, of TCDD (100 fM–10 nM) in DMSO, as well as all solvent
controls. Each sample analysis consisted of nine dilutions of the PCDD/F fraction of eachextract in triplicate, along with appropriate solvent blanks. All materials that came in contact
with TCDD were handled and disposed following procedures approved by the Office ofEnvironmental Health & Safety at the University of California, Davis.
2.4. Statistical methods
General data and statistics were performed using Microsoft Excel. EC50 values from fullconcentration–response curves of the TCDD standard and sample extractions were
determined using the four-parameter Hill equation (SigmaPlot (Systat)), or for incomplete
sample induction curves the EC50 was determined by direct comparison of results to the
TCDD standard curve. EC25 values were determined for those sample concentration
response curves where maximal luciferase activity was less than the EC50 of that of the
TCDD standard curve, by direct comparison to the TCDD standard curve. Total relative
CALUX activity of each extract was determined and expressed as pg bioanalyticalequivalents (BEQs) per gram of soil/sediment.

3. Results and discussion
NIH-PA Author Manuscript

Three main elements are essential for the accurate determination of the relative potency(BEQs) of dl-HAHs present in an extract using the CALUX or any other AhR-based
bioassay: (1) efficient extraction and clean-up procedures to isolate the desired dl-HAHs
from the sample extracts with little carry over of undesired AhR agonists; (2) specificcriteria establishing comparisons of sample extract and TCDD concentration induction
curves; and, (3) data covering the best possible range of induction in order to establish ascomplete a sample extract induction curve as possible with the optimal results having bothmaximal and minimal plateaus to the induction curve and a slope comparable to that of the
TCDD standard curve. In CALUX analysis, decreasing amounts of sample extract are added
to the cells and if AhR active compounds or dl-HAHs are present, a concentrationdependent luciferase induction response curve is generated that can be directly compared tothat produced by increasing concentrations of TCDD, thus allowing calculation of therelative inducing potency of the sample extract after correction for sample dilution [14,19].
While relative potency values estimated from GC/HRMS analysis are expressed as TEQ
values (utilizing TEFs obtained from in vivo toxicity studies [10]), relative potency values

Talanta. Author manuscript; available in PMC 2012 February 15.

Baston and Denison

Page 5

NIH-PA Author Manuscriptfrom our CALUX bioassay luciferase induction results are expressed as BEQs. Use of BEQ
is more appropriate and less confusing than the range of previously reported bioanalyticalpotency descriptors such as: TEQ, TCDD equivalents, Bio-TEQs, CALUX-TEQs, IEQs
(induction equivalents) and others, especially since AhR-based bioassays do not provide a
direct measure of the toxicity of dl-HAHs present in the mixture [19], but only of its potency
to activate the AhR and AhR-dependent gene expression (which is mechanistically related tothe adverse effects of dl-HAHs [1,2,5–10]).

NIH-PA Author Manuscript
NIH-PA Author Manuscript

While assessment of the BEQs of sediment/soil extracts by CALUX bioassay analysis has
been standardized, there is significant diversity in actual concentration–response to different
sample extracts and this can impact both data interpretation and the accuracy of potency(BEQ) determinations [14,19,32,39]. Accordingly, we have examined a wide variety of
sediment/soil samples by CALUX analysis in order to present the actual diversity inresponse observed for this assay and approaches taken to improve bioassay-based potency
determination and assay interpretation. Incubation of the CALUX cells with TCDD results
in a concentration-dependent induction of luciferase activity significantly above background
beginning at a concentration of ~0.1 pg/well to a maximal activity at ~100 pg/well with an
EC50 of ~1–2 pg/well (Fig. 1). Analysis of the PCDD/F fraction of sediment/soil extracts in
the CALUX bioassay resulted in a variety of distinct concentration-dependent luciferase
induction curves with maximal or submaximal induction levels as compared to that of
TCDD (compare Fig. 2 to Fig. 1). While sample 113 induced maximally, and sample 83
induced slightly submaximally, compared to that of TCDD (Fig. 2), the remaining extracts(samples 102, 112, and 118) produced concentration–response curves with the maximal
induction occurring in the linear part of the induction curve, but below TCDD maximal
induction thus yielding an ‘incomplete’ concentration response curve (i.e. a sub-maximal
induction curve). The lower than maximal activity could result from low levels of AhR
agonists in the sample extracts (102, 112 and 118) and the presence of AhR antagonist
which reduce the overall inducing potency of the AhR agonists present in the sample.
Higher concentrations of these extracts could not be evaluated because they produced cell
toxicity (data not shown). These CALUX results are typical of those obtained for soil andsediment extract analysis. Standard approaches for determination of the BEQ from sample
extract concentration induction curves involves using the four-parameter Hill plot todetermine an EC50 value for the TCDD standard curve and for those sample extracts which
produce a full concentration response data (i.e. whose maximal induction activity wascomparable to that of TCDD such as sample 113). However, the four-parameter Hill plotcannot be used when full induction curves (with plateaus at the lower and upper end) are notachieved. For extracts with incomplete or sub-maximal induction yet whose maximal value
exceeds the TCDD EC50 (samples 83 and 112 (Fig. 2)), direct comparison of the EC50s of
the samples to the EC50 for the TCDD standard curve (determined from a four parameter
Hill plot) was used to calculate the BEQs for those sample extracts. In contrast, an EC25
value for TCDD derived using the four parameter Hill plot from the TCDD standard curve,
was used for direct comparison for those extracts whose induction levels were >EC25 but<EC50 of the TCDD standard curve (samples 102 and 118 (Fig. 2)). The results of these
determinations are summarized in Table 1 along with the GC/HRMS-derived TEQ values
for these samples calculated using the WHO 2005 TEF values and previous GC/HRMS
results [38]. Evaluation of the BEQ/TEQ ratio revealed that while CALUX analysis was areasonably good predictor for PCDD/Fs in sample 112 (with a 2-fold higher BEQ value), theBEQs for the remaining sediment samples was between 7- and 266-fold higher than that
determined by GC/HRMS. It is likely that the higher BEQ potency estimate for sample 83
compared to its TEQ value (266-fold higher) results from the fact that the slope of its
induction curve was not parallel to the TCDD standard curve (or that of samples 112 and113), suggesting some modulation (enhancement) of the AhR induction response by anotherchemical(s) present in the extract. Given the uncertainty in BEQ estimates from nonparallel
Talanta. Author manuscript; available in PMC 2012 February 15.

Baston and Denison

Page 6

NIH-PA Author Manuscript

curves, it has been proposed that a range of potency values be determined using various
points on the induction curve in order to better describe the resulting potency estimate. The
elevated BEQ activities of each of the first test set of samples (83,102,112,113,118), relative
to the GC/HRMS TEQ values, suggests that these extracts contain AhR agonists in addition
to the PCDD/Fs. Potential chemicals could include brominated- or mixed chloro/bromodibenzo-p-dioxins, dibenzofurans, chloronaphthalenes and others that have been shown to
be AhR active [31,32,39–42], but are not currently measured using standard GC/HRMS
analysis and currently do not have TEF values.

NIH-PA Author Manuscript
NIH-PA Author Manuscript

Interestingly, concentration-dependent CALUX screening analysis of other sediment andsoil extracts revealed many sample extracts that produced superinduction of luciferase
activity to between 1.5- and 4.5-fold greater than that maximally induced by TCDD (Fig. 3).
While a superinduction response is not entirely surprising given that we have previously
observed this effect using crude soil and sediment sample extracts that have not undergone
any chemical cleanup step to isolate dl-HAHs (data not shown). The fact that a “cleaned-up”
PCDD/F extract fraction can produce this enhanced response suggests that it must containcompounds capable of dramatically enhancing the AhR-dependent CALUX induction
response since superinduction by purified mixtures of PCDD/Fs have not been reported.
Although superinduction of AhR-dependent gene expression has been previously observed
by our laboratory and others [20,33–37], the exact chemical(s) and/or molecularmechanisms responsible have not been elucidated, but several different mechanisms havebeen proposed. Inhibition of AhR degradation by proteolysis has been shown to increase
intracellular levels of ligand (TCDD)-activated AhR and this has been proposed to increase
the magnitude of AhR-dependent gene expression [43,44]. A labile repressor protein has
also been proposed and inhibition of expression and/or enhanced degradation of thisrepressor would result in an increase in AhR functionality and enhanced transcriptional
response [36,43]. Assuming these mechanisms to be responsible for the sediment and soil
extract superinduction response, these extracts would need to contain a chemical(s) thatwould inhibit synthesis of the repressor and/or inhibit cellular proteolytic mechanisms that
degrade the AhR. Although these mechanisms of superinduction are possible, they are
unlikely as these extracts do not produce the toxicity associated with protein synthesis orproteosomal inhibitors. While one might envision that it also may be possible that thesuperinduction response might result from a compound(s) present in these extracts which
has a higher affinity and greater inducing potency than that of TCDD, no such compound
has ever been identified and it is well documented that TCDD is the most potent AhR
agonist [1,2,10]. A more likely explanation is that in addition to containing dioxin-like
HAHs that may co-activate or directly activate the AhR, the extracts also contain achemical(s) that affects other cellular signal transduction pathways augmenting theinduction response. In fact, support for the this hypothesis comes from the observation that
activation of the protein kinase C signaling pathway as well as treatment of CALUX cellswith selected prostaglandins, known signaling factors, results in a synergistic increase in
TCDD-inducible, AhR-dependent gene expression [20,33–37,43,44].
The concentration-dependent superinduction response resulting from a select number ofthese extracts (Fig. 3) revealed that luciferase induction reached a maximal level and plateau
with four of the samples (#95, 96, 97 and 98), while that with samples 94 and 115 failed toreach a maximal level plateau. Similar to the previous results in Fig. 2, it is possible that theincomplete concentration response induction curve derives from the presence of relatively
low levels of AhR agonists in the sample extracts and/or the presence of AhR antagoniststhat reduce the overall inducing potency of the AhR agonists present in the sample.
However, the chemical(s) responsible for the superinduction response would still enhance
the magnitude of induction in either situation. In contrast to the previously establishedapproach to determine the relative potency (BEQ) of the sample extracts shown in Fig. 2,
Talanta. Author manuscript; available in PMC 2012 February 15.

Baston and Denison

Page 7

NIH-PA Author Manuscript
NIH-PA Author Manuscript

estimation of the BEQs from superinduction curves is problematic as these results are notdirectly comparable to the TCDD standard curve. For example, using the standard CALUX
analysis method to directly compare luciferase activity at the EC50 for TCDD to the sameamount of luciferase activity of the superinduced samples is shown in Fig. 4 (compare points
A1 to B1 and C1). Calculation of BEQs using the standard analysis approach and comparing
the values to the GC/HRMS TEQs determined for the same samples (Table 1) reveals thatthe BEQ potency values of the superinduced samples are overestimated by a factor of 5–179-fold. Given our understanding of some of the mechanisms of superinduction of AhR
signaling, it is highly likely that the superinduction response is chemical concentration
dependent and occurs proportionately at all extract concentrations and as such, thesuperinduction response would remain directly proportional to TCDD standard induction.
Accordingly, a more valid approach for relative potency calculation from superinduction
data when a full concentration–response curve is obtained, is to set the maximal induction(i.e. the upper plateau) of the superinduction curve at 100% response and determine the 50%
response (EC50) for each sample curve and then calculate the appropriate BEQ value using
the TCDD EC50 from the standard curve (compare points B2 and C2 to A1 for TCDD (Fig.
4)). This normalization results in a significant decrease (~10-fold) in the overall relative
sample extract potency (BEQ value) of the superinducing sample extract compared to that
estimated by direct comparison of results with the EC50 of the TCDD standard curve (Fig.
4, compare points B1 to B2). Alternatively, for visual clarity and comparative purposes(both for potency and to confirm parallel induction responses), the results of each curve canbe normalized to its own maximal activity and all of the results plotted together (Fig. 5).
Comparison of the BEQ values for the four superinducing sediment samples that produced
full concentration response curves (samples 95–98) before and after normalization (Table 1),
not only revealed that normalizing the results of these curves decreased their BEQ values by
a factor of 5–9-fold, but the resulting BEQ values were now nearly identical to the GC/
HRMS TEQ values (BEQ/TEQ ratio of 0.7–1). Thus, internal normalization of CALUX
superinduction results from full concentration–response curves allows increased accuracy
and precision of relative potency estimates of PCDD/Fs in sample extracts.

NIH-PA Author Manuscript

Full concentration response curves are not obtained for most sample extracts run in the
CALUX bioassay and while this can complicate BEQ estimations from these types of data,
the accuracy of the BEQ estimate depends on the magnitude of the induction response
obtained (i.e. do the maximal results fall in the linear working range and/or are they greaterthan 50% of the maximal TCDD induction response). This is even more problematic whenattempting to calculate BEQs from superinduction results of concentration response curvesof submaximal induction curves (i.e. those that do not reach an upper plateau (Fig. 4C)),
since the maximal response we would set to 100% is unknown. Accordingly, the mostconservative approach for BEQ estimation from submaximal superinduction results would
be to set the highest point of the induction curve as the maximal induction (i.e. 100%
response) and arithmetically determine the 50% value (EC50) of the induction curve (Fig.
4C). The adjusted decrease in extract potency is demonstrated by a nearly 10-fold increase
in EC50 potency value, compare points C1 and C2 in Fig. 4, and while it is acknowledgedthat there may be some overestimation of the overall sample extract potency, the resulting
BEQ value from this internal normalization is closer to the analytical toxic potency (i.e. theTEQ). Normalization of the sub-maximal superinduction results to 100% response also
allows them to be directly compared to all other CALUX concentration–response curves(Fig. 5). Direct comparison of the effect of normalization of submaximal superinduction
data (samples 94 and 115) reveal that normalization significantly decreases the CALUX
BEQ potency estimate and increases the accuracy of the BEQ estimate of the PCDD/F TEQ
values for these sample extracts by 8–13-fold (i.e. reducing the standard analysis BEQ/TEQ
ratio for samples 94 and 115 from 179 and 74, respectively, to 21 for both samples withnormalization (Table 1)). The lack of a complete induction curve for the submaximal
Talanta. Author manuscript; available in PMC 2012 February 15.

Baston and Denison

Page 8

NIH-PA Author Manuscript

superinducing sample extracts certainly contribute to the remaining overestimation of thepotency of these samples, although the impact of other factors such as the presence ofadditional AhR agonists in these extracts cannot be discounted. Taken together, our results
demonstrate that normalization of sample extract CALUX superinduction results provides avalid approach for more accurate determination of BEQs of dl-HAHs in sample extracts andshould be used when such a response is obtained.

NIH-PA Author Manuscript

The improved correlation between TEQs and normalized superinduction BEQs, coupledwith our relatively common observation of CALUX superinduction by soil and sediment
samples, suggests that evaluating soil and sediment extract potencies by simple directcomparison to the EC50 of the TCDD standard curve without full concentration–response
analysis can contribute to overestimated CALUX-based BEQ values. Superinduction could
even contribute to BEQ overestimation for those sample extracts that to not induce to 100%of that maximally induced by TCDD. In this instance, the presence of “superinducing”
compounds in the sample extract could enhance the magnitude of response to a low level of
AhR-active dl-HAHs, thus making the sample appear more potent than it actually is andresulting in an overestimated BEQ value. However, until the mechanisms responsible for thesuperinduction responses by these sediment/soil and other extracts are elucidated, thecontribution of superinduction to BEQ potency estimates from submaximal induction curves
cannot be determined. Taken together, our results not only report for the first time thephenomenon of superinduction of the CALUX bioassay by “cleaned-up” PCDD/F fractionsof sediment and soil extract, but we also provide an avenue by which normalization of suchdata leads to more accurate potency estimates. In addition, they demonstrate that for optimaland most accurate BEQ determination for all soil, sediment, or environmental extracts by
CALUX and likely other AhR-based bioassays, whenever possible, full concentration
response curves should be utilized.

Acknowledgments
We thank Dr. Steven Safe for the TCDD. We acknowledge support from a National Institutes of Environmental
Health Sciences (NIEHS) Superfund Research Grant [P42ES004699], the California Agricultural Experiment
Station and the American taxpayers.

References

NIH-PA Author Manuscript

1. Poland A, Knutson JC. Annu Rev Pharmacol Toxicol. 1982; 22:517–554. [PubMed: 6282188]
2. Safe S. S Crit Rev Toxicol. 1990; 21:51–88.
3. DeVito, MJ.; Birnbaum, LS. Dioxins and Health. Schecter, A., editor. Plenum Press; New York:1994. p. 139-162.
4. Giesy, JP.; Ludwig, JP.; Tillitt, DE. Dioxins and Health. Schecter, A., editor. Plenum Press; New
York: 1994. p. 254-307.
5. Denison, MS.; Elferink, CF.; Phelan, D. Toxicant–Receptor Interactions in the Modulation of Signal
Transduction and Gene Expression. Denison, MS.; Helferich, WG., editors. Taylor & Francis;
Bristol, PA: 1998. p. 3-33.
6. Bradshaw TD, Bell DR. Clin Toxicol. 2009; 47:632–642.
7. Furness SG, Whelan F. Pharmacol Ther. 2009; 124:336–354. [PubMed: 19781569]
8. Denison MS, Nagy SR. Annu Rev Pharmacol Toxicol. 2003; 43:309–334. [PubMed: 12540743]
9. Mitchell KA, Elferink CJ. Biochem Pharmacol. 2009; 77:947–956. [PubMed: 19027718]
10. van den Berg M, Birnbaum LS, Denison MS, De Vito M, Farland W, Feeley M, Fiedler H,
Hakansson H, Hanberg A, Haws L, Rose M, Safe S, Schrenk D, Tohyama C, Tritscher A,
Tuomisto J, Tysklind M, Walker N, Peterson RE. Toxicol Sci. 1996; 93:223–241. [PubMed:
16829543]
11. Liem AKD. Trends Anal Chem. 1999; 18:429–439.

Talanta. Author manuscript; available in PMC 2012 February 15.

Baston and Denison

Page 9

NIH-PA Author Manuscript
NIH-PA Author Manuscript
NIH-PA Author Manuscript

12. Liem AKD. Trends Anal Chem. 1999; 18:499–507.
13. Behnisch PA, Hosoe K, Sakai S. Environ Int. 2001; 27:413–439. [PubMed: 11757855]
14. Whyte JJ, Schmitt CJ, Tillitt DE. Crit Rev Toxicol. 2004; 34:1–83. [PubMed: 15000436]
15. Denison, MS.; Rogers, JM.; Rushing, SR.; Jones, CL.; Tetangco, SC.; Heath-Pagliuso, S. Current
Protocols in Toxicology. Maines, M.; Costa, LG.; Reed, DJ.; Sassa, S.; Sipes, IG., editors. John
Wiley and Sons; New York: 2002. p. 4.8.1-4.8.45.
16. Giesy JP, Hilscherova K, Jones PD, Kannan K, Machala M. Mar Pollut Bull. 2002; 45:3–16.
[PubMed: 12398362]
17. Hahn ME. Sci Total Environ. 2002; 289:49–69. [PubMed: 12049406]
18. Garrison PM, Tullis K, Aarts JMMJG, Brouwer A, Giesy JP, Denison MS. Fundam Appl Toxicol.
1996; 30:194–203. [PubMed: 8812265]
19. Denison MS, Zhao B, Baston DS, Clark GC, Murata H, Han D. Talanta. 2004; 63:1123–1133.
[PubMed: 18969542]
20. Seidel SD, Winters GM, Rogers WJ, Ziccardi MH, Li V, Keser B, Denison MS. J Biochem Mol
Toxicol. 2001; 15:187–196. [PubMed: 11673847]
21. Han D, Nagy SR, Denison MS. Biofactors. 2004; 20:11–22. [PubMed: 15096657]
22. Denison, MS.; Seidel, SD.; Rogers, WJ.; Ziccardi, M.; Winter, GM.; Heath-Pagliuso, S. Molecular
Biology Approaches to Toxicology. Puga, A.; Wallace, KB., editors. Taylor & Francis;
Philadelphia: 1998. p. 393-410.
23. Denison MS, Heath-Pagliuso S. Bull Environ Contam Toxicol. 1998; 61:557–568. [PubMed:
9841714]
24. Nagy SR, Liu G, Lam K, Denison MS. Biochemistry. 2002; 41:861–868. [PubMed: 11790108]
25. Khim JS, Villeneuve DL, Kannan K, Koh CH, Giesy JP. Environ Sci Technol. 1999; 33:4206–
4211.
26. Tsutsumi T, Amakura Y, Nakamura M, Brown DJ, Clark GC, Sasaki K, Toyoda M, Maitani T.
Analyst. 2003; 128:486–492. [PubMed: 12790202]
27. Van Wouwe N, Windal I, Vanderperren H, Eppe G, Xhrouet C, De Pauw E, Goeyens L, Baeyens
W. Talanta. 2004; 63:1269–1272. [PubMed: 18969557]
28. US EPA Method 4435, Method for Toxic Equivalents (TEQS) Determinations For Dioxin-Like
Chemical Activity with the CALUX Bioassay.
http://www.epa.gov/osw/hazard/testmethods/pdfs/4435.pdf
29. Carbonnelle S, Loco JV, Overmeire IV, Windahl I, Wouve NV, Leeuwen SV, Goeyens L. Talanta.
2004; 63:1255–1259. [PubMed: 18969555]
30. Behnisch PA, Hosoe K, Sakai S. Environ Int. 2003; 29:861–877. [PubMed: 12850102]
31. Olsman H, Engwall M, Kammann U, Klempt M, Otte J, Bavel B, Hollert H. Environ Toxicol
Chem. 2007; 26:2448–2454. [PubMed: 17941736]
32. Schroijen C, Windahl I, Goeyens L, Baeyens W. Talanta. 2004; 63:1261–1268. [PubMed:
18969556]
33. Chen YH, Tukey RH. J Biol Chem. 1996; 271:26261–26266. [PubMed: 8824276]
34. Long WP, Pray-Grant M, Tsai JC, Perdew GH. Mol Pharmacol. 1998; 53:691–700. [PubMed:
9547360]
35. Long WP, Chen X, Perdew GH. J Biol Chem. 1998; 274:12391–12400. [PubMed: 10212212]
36. Joiakim A, Mathieu PA, Elliott AA, Reiners JJ Jr. Mol Pharmacol. 2004; 66:936–947. [PubMed:
15385644]
37. Ma Q. Arch Biochem Biophys. 2002; 404:309–316. [PubMed: 12147270]
38. Nichkova M, Park EK, Koivunen ME, Kamita SG, Gee SJ, Chuang J, Emon JM, Hammock BD.
Talanta. 2004; 63:1213–1223. [PubMed: 18969550]
39. Windahl I, Denison MS, Birnbaum LS, van Wouwe N, Baeyens W, Goeyens L. Environ Sci
Technol. 2005; 39:7357–7364. [PubMed: 16245802]
40. Villeneuve DL, Kannan K, Khim JS, Falandysz J, Nikiforov VA, Blankenship AL, Giesy JP. Arch
Environ Contam Toxicol. 2000; 39:273–281. [PubMed: 10948276]

Talanta. Author manuscript; available in PMC 2012 February 15.

Baston and Denison

Page 10

NIH-PA Author Manuscript

41. Puzyn T, Falandysz J, Jones PD, Giesy JP. J Environ Sci Health A: Tox Hazard Subst Environ
Eng. 2007; 42:573–590. [PubMed: 17454365]
42. Samara F, Gullett BK, Harrison RO, Chu A, Clark GC. Environ Int. 2009; 35:588–593. [PubMed:
19121869]
43. Ma Q, Baldwin KT. Chemosphere. 2002; 46:1491–1500. [PubMed: 12002481]
44. Ma Q, Renzelli AJ, Baldwin KT, Antonini JM. J Biol Chem. 2000; 275:12676–12683. [PubMed:
10777561]

NIH-PA Author Manuscript
NIH-PA Author Manuscript
Talanta. Author manuscript; available in PMC 2012 February 15.

Baston and Denison

Page 11

NIH-PA Author Manuscript
NIH-PA Author Manuscript

Fig. 1.

Concentration–response curve for the induction of luciferase activity by TCDD in CALUX
H1L6.1c3 cells. Cells were plated in 96-well plates for 24 h and incubated with the indicatedconcentration of TCDD (pg/100 μl incubation volume) for 24 h, followed by determinationof luciferase activity as described in Section 2. Values were expressed as a percent of the
maximum induction by TCDD and represent the mean ± SD of at least triplicate
determinations.

NIH-PA Author Manuscript
Talanta. Author manuscript; available in PMC 2012 February 15.

Baston and Denison

Page 12

NIH-PA Author Manuscript
NIH-PA Author Manuscript

Fig. 2.

Concentration-dependent induction of luciferase activity by extracts of soil/sediment in
CALUX mouse H1L6.1c3 cells. Cells were plated and incubated with increasing
concentrations of the PCDD/F fraction of the indicated soil/sediment extracts (mgequivalents/100 μl incubation volume) and luciferase activity determined as described in
Section 2. Values were expressed as a percent of the maximum induction by TCDD andrepresent the mean ± SD (n ≥ 3).

NIH-PA Author Manuscript
Talanta. Author manuscript; available in PMC 2012 February 15.

Baston and Denison

Page 13

NIH-PA Author Manuscript
NIH-PA Author Manuscript

Fig. 3.

Superinduction of luciferase by extracts of soil/sediment in CALUX mouse H1L6.1c3 cells.
Cells were plated and incubated with increasing concentrations of the PCDD/F fraction of
the indicated soil/sediment extracts (mg equivalents/100 μl incubation volume) andluciferase activity determined as described in Section 2. Values were expressed as a percentof the maximum induction by TCDD and represent the mean ± SD (n ≥ 3).

NIH-PA Author Manuscript
Talanta. Author manuscript; available in PMC 2012 February 15.

Baston and Denison

Page 14

NIH-PA Author Manuscript
Fig. 4.

Approach to determination of relative potency values (EC50) values for soil/sediment
samples producing superinduction in the CALUX bioassay (see text for details).

NIH-PA Author Manuscript
NIH-PA Author Manuscript
Talanta. Author manuscript; available in PMC 2012 February 15.

Baston and Denison

Page 15

NIH-PA Author Manuscript
NIH-PA Author Manuscript

Fig. 5.

Normalization of concentration-dependent superinduction of luciferase by extracts of soil/
sediment in CALUX mouse H1L6.1c3 cells. CALUX induction results for soil/sedimentextracts in Fig. 3 were each normalized to 100% of their respective maximal induction leveland expressed as the mean ± SD (n ≥ 3) of the percent of the maximal value.

NIH-PA Author Manuscript
Talanta. Author manuscript; available in PMC 2012 February 15.

NIH-PA Author Manuscript

NIH-PA Author Manuscript
68
43
8582
8397
10968
10773
13

118

94

95

96

97

98

115

SI-SM

SI-F

SI-F

SI-F

SI-F

SI-SM

S50

F

SM

S50

F

Curve typea

958

71244

57654

70985

43309

7679

NA

28317

24

NA

ND

ND

ND

ND

ND

ND

484

ND

ND

864

c
Not applicable.

Not determined.

b

74

7

5

8

5

179

7

56

2

54

266

BEQ/TEQ

275

8500

7472

7680

8726

896

NA

NA

NA

NA

NAc

BEQ (nEC50) (pg eqv/g)

NDb

BEQ (EC25) (pg eqv/g)

BEQ (EC50) (pg eqv/g)
60918

Normalization

Standard analysis

Full curve (F), submaximal (SM), sub-EC50 (S50), superinduction full curve (SI-F), superinduction submaximal (SI-SM).

a

13
502

16

102

113

229

83

112

GC/MS TEQs (pg/g sed)

Sample no.

21

0.8

0.7

0.9

1

21

NA

NA

NA

NA

NA

BEQ/TEQ

Summary of CALUX BEQ and GC/HRMS TEQ determination for extracts of soil and sediment. Soil and sediment samples were extracted and analyzed
using standard methodology by CALUX and GC/HRMS. For extracts that superinduced in the CALUX bioassay (SI-F and SI-SM), each concentration–
response curve was normalized to its maximal induction level (set at 100%) and EC50 values for each curve determined as described in the text.

NIH-PA Author Manuscript

Table 1
Baston and Denison
Page 16

Talanta. Author manuscript; available in PMC 2012 February 15.