SUPERINDUCTION OF THE CALUX BIOASSAY BY ENVIRONMENTAL EXTRACTS:CONSIDERATIONS IN SAMPLE ANALYSIS AND POTENCY DETERMINATIONDavid S. Baston1, Elaine M. Khan1 and Michael S. Denison1
1. Department of Environmental Toxicology, Meyer Hall, University of California, Davis, CA 95616.
Introduction
Accurate evaluation of environmental samples for the presence of anthropogenic contaminants continues to be
an important aspect of determining the human risk potential from exposure to these materials. While highresolution instrumental methods (i.e. GC/HRMS) are the gold standard for identification and quantitation of
halogenated aromatic hydrocarbons (HAHs), such as polychlorinated dioxins, furans and biphenyls, numerous
rapid and relatively inexpensive screening bioanalytical methods have been developed.1 The Chemically
Activated LUciferase eXpression (CALUX) cell bioassay is one technology that has been extensively used forthe detection and relative quantitation of HAHs in a wide variety of environmental, biological and food matrices
and is based on the ability of these chemicals to bind and activate the Ah receptor (AhR) and AhR signaltransduction (i.e. gene expression).1 The CALUX cell lines contain a stably transfected AhR-responsive firefly
luciferase reporter gene that responds to dioxin (2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD, dioxin)) andrelated dioxin-like chemicals with the induction of firefly luciferase gene expression in a chemical-, dose-, timeand AhR-dependent manner. The CALUX bioassay provides an avenue to determine the relative quantity ofAhR-active HAHs present in a given sample extract, relative to that of TCDD and this is typically accomplishedby comparing the concentration-response curve for luciferase induction by the sample extract (containing acomplex mixture of chemicals) directly to that obtained with increasing concentrations of the TCDD standard.
Direct comparison between curves allows calculation of a Bioanalytical Equivalent (BEQ) (i.e., a luciferase
induction equivalent) for the sample extract relative to that of TCDD dose curve. From a statistical perspective,
comparison of the effective concentration at 50% induction (EC50) of each induction curve (the point at which
luciferase induction is 50% of maximal) provides the most accurate measurement. However, accurate
determination of BEQs from direct comparisons of sample extract and TCDD standard curves requires that thesample extract concentration curves have similar characteristics to that of the TCDD standard curve (i.e. same
slope and minimum and maximal induction activity), which would be expected since both utilize the AhR
induction mechanism. However, given the nature of the biological CALUX cell system, it is not surprising thatextracts containing complex mixtures of chemicals can produce CALUX induction curves that deviate from thatof the TCDD standard and numerous methods for estimating sample extract potency from dose curves notmeeting these criteria have been reported.1,2 During our extensive analyses of environmental samples by
CALUX, we have found numerous samples that induce CALUX luciferase gene expression to levels 2-3 times
greater than that induced by a maximal inducing concentration of TCDD. This superinduction phenomenon
complicates accurate estimation of the TCDD BEQs for a given sample extract and can result in overestimationof the potency if standard comparative analysis of induction curves are performed. Here we describe andcharacterize the CALUX superinduction phenomenon using sediment extracts and provide an method to moreaccurately calculate the BEQs for sample extracts that produce this response.
Methods
Sample Extract Preparation. Dry, crushed sediment, soil, and plant samples were acquired from ourcollaborators. Extracts were prepared in hexane or ethanol from 2 g of sample. For analysis of HAHs, sample
extracts were cleaned up by filtering them through a sodium sulfate/celite column, followed by a 33% acid silica
column and fractionated on a 1% X-CARB column.3 To analyze crude sample extracts (without cleanup),extracts were only filtered through a sodium sulfate/celite column and the sample/column eluate collected. The
final extract volume for each sample was ~4 ml of either hexane or ethanol. Prior to analysis, aliquots of eachsample extract (crude or cleaned-up) were dried and resuspended in a small volume of DMSO or ethanol(ETOH) and tested in the CALUX bioassay as described.3,4Cell Culture, Chemical Treatment and CALUX Analysis. Stably transfected CALUX cell lines, H1L6.1c3 (forAhR analysis) and BG1Luc4E2 (for xenoestrogen analysis) were grown as described. The inducing potency ofOrganohalogen Compounds, Volume 70 (2008) page 000406

each sample was evaluated in a range finding study, followed by a nine-point dose curve.4-6 Cells in a 96-well
microplate format were incubated with standards or extracts for 24 hours and luciferase activity determinedusing a Anthos Lucy 2 or Berthold microplate luminometer as described.
Results and DiscussionCALUX is an AhR-based cell bioassay system that responds to TCDD and related TCDD-like HAHs with theinduction of firefly luciferase reporter gene expression. Exposure of these cells to TCDD or extracts containing
these chemicals or other AhR agonists results in a typical receptor-dependent sigmoidal concentration (dose)response curve, from which the relative inducing potency (i.e. BEQ) of the test extract or chemical can becalculated when compared to the TCDD standard curve. Determination of the relative potency of sample
extracts or chemicals that produce a full concentration-response curve in the CALUX bioassay with a maximal
activity and slope comparable to that produced by TCDD are simple to calculate by direct comparison of theEC50s of the curves (Figure 1; compare TCDD (point 1) and sample C (point 4)) and many linear curve fitting
analyses such as the 4-parameter Hill plot or probit analysis have been used.1,2 However, sample extracts or
chemicals that fail to produce a full concentration-response curve comparable to that of TCDD, or theconcentration response curve plateaus at a level significantly lower than that produced by TCDD (typified bypartial agonists) or those whose slope of the concentration-response curve deviates significantly from that
produced by TCDD are commonly encountered and determination of the relative potency of these sample
extracts or chemicals are more complicated.1,2 Several approaches have been described to attempt to determine
the relative potency of these types of samples and unlike the full response curve results, these latter relative
potency value estimates are less certain. During our extensive extract screening analysis of environmental,biological and food samples, as well as pure chemicals, each of these types of concentration response curves
have been observed and we have evaluated and selected the mathematical approaches that lead to the mostaccurate and justifiable estimate of their relative potency. Our screening results also allowed identification ofnumerous sample extracts that when tested in the CALUX bioassay resulted in a concentration-response curve
whose maximal luciferase activity was significantly greater than that of the TCDD standard curve.
Determination of the relative potency of these "superinducing" extracts is particularly problematic given that
there exist no published methodology to use to calculate/estimate potency of samples that induce more than themaximal activity of the most potent AhR agonist (TCDD). To examine this CALUX "superinduction"
phenomenon in greater detail and to determine an appropriate method to calculate relative potencies for theseextracts, we selected sediment sample extracts that induced CALUX luciferase activity to varying degrees.
These included an extract that induced CALUX luciferase activity to the same maximal activity as that ofTCDD (Figure 1, sample C) and two sample extracts that could "superinduce" CALUX luciferase reporter geneactivity (one that produced a full concentration-response curve (Figure 1, sample B) and one that produced asubmaximal concentration-response curve (Figure 1, sample A)). Sediment sample A and B induced CALUX
luciferase gene expression to a level ~2- and 3-times greater than a maximal inducing concentration of TCDD,respectively. A common approach to estimate the relative potency for an unknown sample extract is to directlycompare the luciferase activity at the EC50 for TCDD to the identical luciferase activity induced by the sample
extract and using the concentration of TCDD at the EC50 to estimate the relative concentration of TCDD
bioanalytical equivalents that must be present in the sample extract in order to generate the comparable
induction response. While this is an appropriate approach for sample extracts whose induction curves areparallel to and reach the same maximal as the TCDD standard curve, it is not necessarily appropriate for thosesamples whose maximal CALUX induction activity is less than or greater than the maximal activity induced byTCDD. For example, estimation of the relative potency of sample C (5.2 ng TCDD equivalents/g sediment)using the TCDD standard curve would be appropriate since it meets all established criteria (Figure 1 [comparepoints 1 and 4] and Table 1). However, estimation of the relative potency of sample A (point 2) and B (point 3)
using this same direct EC50 luciferase activity comparison approach results in 5.4 and 209 ng TCDD
equivalents/g sediment, respectively. If we consider that the synergistic enhancement of the CALUX geneinduction response is likely occurring across the entire concentration range, combined with the fact that both
induction responses use the exact same receptor (i.e. the AhR), it is justified to normalize or scale thesuperinduction curves to a maximum of 100% relative to that of TCDD. For sample B, this is simple since a fullconcentration-response curve was obtained and recalculation of the relative potency of this extract using its
actual EC50 (point 5 in Figure 1) to that of TCDD, results in a BEQ of 29.7 ng TCDD equivalents/g sediment, aOrganohalogen Compounds, Volume 70 (2008) page 000407

7-fold decrease in the estimated potency compared to the previous calculation (Table 1). In contrast, thesuperinduction response with sample A does not reach a maximal activity, so the concentration curve can't
simply be scaled/normalized to that of TCDD. In this instance, we propose to use the highest induction
response value for nonmaximal superinduction curves as the 100% response value and the concentration
response curve scaled as described for sample B. While the EC50 value and BEQ generated using this approach
would overestimate sample potency, since maximal activity was not obtained, the BEQ estimate resulting fromthis scaled determination would still be closer to the actual potency value. Using this approach, the relative
potency (BEQ) for sample A was calculated to be 1.2 ng TCDD equivalents/g sediment, 4.5-fold less potent
than that calculated using the direct TCDD EC50 comparison (Table 1). The observation of superinduction
responses raises some concerns about the accuracy of relative potency estimations of any sample when thehighest CALUX induction response obtained has not yet reached a maximal (i.e. it is still in the linear portion ofthe curve) and the absolute activity is less than that of the maximal activity of the TCDD standard. If thissample was actually the lower end of a superinduction response curve, the relative potency of the extract (BEQs)
would be greatly overestimated as shown above. The superinduction phenomenon is not unique or an artifact of
the AhR CALUX bioassay, as we have observed a similar superinduction responses using a stably transfectedestrogen receptor (ER)-responsive luciferase reporter gene in human ovarian carcinoma cells (BG1) following
exposure to crude solvent extracts of plants (data not shown). While it remains to be determined what accounts
for the superinduction response to the sediment extracts in the CALUX cell line, we and others have observedthat co-treatment of CALUX cells with TCDD and a chemical (phorbol ester) that stimulates protein kinase C
activity will result in a synergistic increase in AhR-dependent gene expression (Figure 2).6,7 One can envisionthat substances in the complex mixture of chemicals in an extract could stimulate the AhR (and ER) and protein
kinase C, leading to enhanced transcriptional activation activity of the these receptors and/or basal transcription
machinery. While the exact mechanism of superinduction is unknown, further investigation may allow
identification of a step that can be incorporated into the standard CALUX bioassay protocol to significantly
increase assay sensitivity and response. Such studies are underway.
References:
1. Denison MS, Zhao B, Baston DS, Clark GC, Murata H, Han, D-H (2004) Talanta 63:1123.
2. Whyte JJ, Schmitt CJ, Tillitt DE (2004) Crit Rev Toxicol 34:1.
3. Brown DJ, Orelien J., Gordon JD, Chu AC, Chu MD, Murata HJ, Kayama F, Denison MS, Clark GC (2007)
Environ Toxicol Chem 41:4354.
4. Rogers JM, Denison MS (2000) In Vitro Molec Toxicol 13:67.
5. Ziccardi MH, Gardner IA, Denison MS (2000) Toxicol Sci 54:183.
6. Chen Y-H, Tukey RH (1996) J Biol Chem 271:26261.
7. Long WP, Chen X, Perdew GH (1998) J Biol Chem 274:12391.
Table 1. Relative potency comparison of sediment extracts. Results were calculated relative
to the TCDD EC50 or scaled values from the superinduction curves. See text for details.
BEQ Values
EC50 Values(ng TCDD Eqv. /g Sediment)
Direct TCDD
Superinduced
Direct TCDD
Superinduced
Sample Curves
EC50 Comparison
Curve
EC50 Comparison
Curve
TCDD Standards
4.049 (1)a(pg)Sample A Eq. (g)0.75E-3 (3)3.38E-3 (6)

5.39

1.20

Sample B Eq. (g)0.019E-3 (2)0.14E-3 (5)208.93

29.66

Sample C Eq. (g)0.76E-3 (4)-5.31-a. Numbers refer to calculated points in figure 1.

Organohalogen Compounds, Volume 70 (2008) page 000408

Figure 1. Concentration-response curves for TCDD and sediment extracts. The numbers indicate the EC50s of
the induction curves relative to TCDD (points 1-4) or relative to the superinduction curve (points 5 and 6).
32

10-1-1-1-1-9-8-7-6-5-4-3-2-11e1e1e1e1e1e1e1e1e1e1e1e1e1e+14-1

1e+05-11e6-11e

350

1e

TCDD Concentration (M)TCDD Conc (M)Sample A (g)300

Sample B (g)Sample C (g)% TCDD Maximum

250

200

5150

6100

2

1350

4-2-1

1e

1e+1-31e1e-4
1e+0-5
1e

1e-6
1e0-1

1e-7

1-1

1e-82-11e1e3-11e1e4-11e-95-1

1e1e6-1
1e0Sediment Equivalents (g)Figure 2. Stimulation of protein kinase C with aphorbol ester (PMA) synergistically increases
TCDD-dependent induction of luciferase
activity in the CALUX cell bioassay.

Organohalogen Compounds, Volume 70 (2008) page 000409