BIOANALYSISELIMINATION OF INTERFERING COMPOUNDS IN PREPARATIONFOR ANALYSIS BY AN Ah RECEPTOR BASED BIOASSAYDavid J. Brown1, Ilse Van Overmeire2, Leo Goeyens2, Michael D. Chu 1, Michael S. Denison 3 andGeorge C. Clark1
1

Xenobiotic Detection Systems, Inc., 1601 East Geer Street, Suite S, Durham, NC 27704, USA
Department of Pharmaco-Bromatology, Scientific Institute of Public Health, Juliette Wytsmanstraat14, 1050 Brussels, Belgium
3
Department of Enviromental Toxicoogy, 4241 Meyer Hall, University of California, Davis, CA 95616,USA2Introduction
In general it has been assumed that bioassays are less sensitive to sample clean up than chemical analysis
methods. For chemical analysis methods such as gas chromatography/mass spectrometry (HRGC/HRMS) it
is necessary to submit samples to extensive and expensive clean up in order to remove compounds that willinterfere with the measurement of the compounds of interest. This is especially true for biological samples
where compounds such as lipids must be removed to avoid compromising the performance of theinstrument. In contrast bioassays tend to be more robust in relation to biological compounds and it is notnecessary to remove these compounds in sample clean up. As long as the sample extract does not containcompounds that are acutely toxic to the bioassay, the results should indicate the presence of compounds thatbind to and activate the Aryl hydrocarbon receptor (AhR). Concerns about sample clean up for bioassays
arise when it is considered that bioassays can respond to a wide range of different compounds. Unlike
chemical analysis, which measures the concentrations of specific compounds, bioassays sum the affects of
all compounds present in the sample extract that can bind to and activate the AhR. For a crude extract thismay include both “classical” and “non-classical” AhR ligands as described by Denison et al.1 “Classical” Ahligands are hydrophobic compounds that have a planar, aromatic structure similar to that of 2,3,7,8tetrachlorodibenzo-p-dioxin (TCDD). The “classical” AhR ligands include the polyhalogenateddibenzodioxins/dibenzofurans (PHDD/F), polyhalogenated biphenyls (PHB), polybrominated diphenylethers (PBDE), as well as some other polyaromatic hydrocarbons (PAH). Other compounds have beenidentified that can bind to and activate the AhR, but do not have the same structural characteristics as the“classical” AhR ligands. These “non-classical” AhR ligands include biological molecules such as bilirubin,
tryptophan metabolites, and some members of the corticosteroid group.
AhR based bioassays (AhR binding or gene expression assays) do not discriminated between thesevarious active compounds. Therefore, it is necessary to use sample clean up as a way to isolate thecompounds that are of interest. Generally, this involves the isolation of PHDD/F and PHB, The twogroups of “classical” AhR ligands that are currently widely regulated. Here we report on various
sample preparation methods and their efficacy at removing environmental contaminants (pesticides andPAH) as well as selected “non-classical” AhR ligands.
Materials and Methods
Materials
Compounds screened for CALUX activity were purchased from Sigma-Aldrich, Inc. and
ChemServices, Inc. TCDD was purchased from Wellington Laboratories.

ORGANOHALOGEN COMPOUNDS Vol. 58 (2002)

401

BIOANALYSISCALUX Assay
XDS has developed a cell line (mouse hepatoma H1L6.1) that was stably transfected with a vector
that contains the gene for firefly luciferase under transactivational control of the aryl hydrocarbon
receptor2. Serial dilutions of the compounds of interest were prepared in dimethyl sulfoxide. Prior to
dosing the cells, the DMSO solutions were suspended in cell culture medium and the medium added tomonolayers of the cells grown in 96 well culture plates. In addition to the samples, a standard curve ofTCDD was assayed (a two-fold dilution series beginning at 1.55 x 10 -9 M TCDD). The plates wereincubated for 20 hours at 37 oC in a humidified CO 2 incubator to allow optimal luciferase geneexpression. Following incubation, the medium was removed and the cells were examined
microscopically for viability. The induction of luciferase activity was quantified using the luciferase
assay kit from Promega (Madison, WI).
Identification of interfering compounds
More than two hundred compounds were tested in the CALUX assay for activity. A ten-folddilution series of each compound starting at 25 milligram/ml DMSO (or highest concentration that wassoluble) was tested. It was necessary to test a series of dilutions as some compounds were acutely toxic
to the cells and could have resulted in a false negative result if only tested at the highest concentration.
Compounds that were active were further submitted to a two-fold dilution series and the CALUX TEQ/
mg compound were determined.
Removal of potential interfering compounds by sample clean up
Active compounds were prepared as a hexane solution and one milligram of the compound wassubmitted to three different sample clean up methods.
Acid silica only treatment: a hexane solution of the compound was passed over a hexane rinsed 33% (w/w) sulfuric acid:silica gel column and the eluate was collected in a hexane rinsed glass tube.
Following sample addition, the column was rinsed with three column volumes of hexane and thepooled eluate was concentrate under vacuum until nearly dry. The residue was resuspended to four
milliliters with hexane and a portion of this was analyzed for activity in the CALUX assay.
Acid silica/XCARB treatment: An acid silica column was prepared as describe above and placed
above an XCARB column containing activated carbon. The compound in a hexane solution was added
to the acid silica column and the column was eluted as described above. The acid silica column wasremoved and the XCARB column was rinsed with hexane and then differentially eluted to yield twofractions. The first collected fraction contained PHB and the second fraction contains PHDD/F.
Results and Discussion
Identification of interfering compounds and removal by sample cleanup
After screening over two hundred compounds, 10 were identified that activated the CALUX assay
and were potential interfering compounds. Figure 1 shows representative dose response curves forthree of the compounds that were tested. Inactive compounds were defined as having maximal activityof less than 10 % of the TCDD maximal response or no clear dose response (o-cresol). Acute toxicityof the compound (acenaphylene) or precipitation of the compound at the highest concentrations(chrysene) often made it difficult to measure a complete dose response curve. Therefore, activity of alltested compounds was measured in the lower half of the dose response curve.
Removal of interfering compounds by sample cleanup
The 10 active compounds were prepared as solutions in hexane and 1 mg of each was submitted toour sample clean up procedure. The resulting fractions were compared to the original solutions to402

ORGANOHALOGEN COMPOUNDS

Vol. 58 (2002)

BIOANALYSIS

Figure 1. Dose response curves for various polyaromatic hydrocarbons.

determine the percent removal of these potential interfering compounds (Table 1). Removal ofactivity varied from treatment to treatment with the highest levels of activity present after acid silica
only (chrysene – 28 % of activity recovered). The acid silica column in series with the XCARB column
provided intermediate results with removal of at least 94 % of activity. This would be sufficient formost samples; however, for samples that are highly contaminated with PAH the sample clean up
method can be exhausted and PAH can make significant contributions to activity in the bioassay. Whenanalyzing sediments from near a coke factory (high PAH levels) we found that greater than 90 % of theactivity of a sample treated by the dual column method and analyzed in the CALUX assay wasassociated with PAH. This may in part explain the finding that the CALUX results tend to be higher
than the corresponding HRGS/HRMS results which have been reported previously3.
It is also important that the sample clean up method be streamlined for high sample throughput.
Bioassays are valuable as a high-throughput screening method and the sample clean up should allow
rapid preparations of samples for analysis. The currently described methods are high throughput
sample clean up methods.
Conclusions
Because AhR based bioassays sum the affects of all active compounds that are present, it isnecessary to use sample clean up as a means of isolating the compounds that are of interest. The resultspresented here indicate that it is possible to remove interfering compounds that are AhR active, but are
not PHDD/F or PHB. The choice of sample clean up method used can have a significant impact on theamount of activity from these interfering compounds that survives the clean up process and therefore
can have an impact on the reported TEQ determination for the sample.

ORGANOHALOGEN COMPOUNDS Vol. 58 (2002)

403

BIOANALYSIS
Table 1. Recoveries of pure compounds submitted to sample clean up method. Dioxin fraction andPCB fraction were the respective fractions from an acid silica column and XCARB carbon column inseries. Percent recoveries reported as the percent of the original activity applied to the clean up method.
ND = no activity detected following clean up.
Compoundbenzo(a)pyrene
chryseneacenaphthylene
benzo(a)anthracene
benzo(b)fluoranthenebenzo(k)fluoranthenecreosotep-cresoldibenzo(a,h)anthracene
1,2-diphenylhydrazine
fluorene
ideno(1,2,3 cd)pyrene
perylene
2-phenylindoleo-cresol
tryptamine

Acid silica only
% recovery

Dioxin fraction% recovery

PCB fraction% recovery

0.002 %
28 %ND
0.05 %
26 %<0.001 %ND
ND<0.001 %
ND
ND
0.6 %
ND
ND
ND
ND

0.02 %
0.2 %0.2 %0.005 %
5.7 %
<0.001 %
ND
ND
ND
ND
ND
2.5 %
ND
ND
ND
ND

ND
ND
ND
ND
<0.001 %
ND
ND
ND
ND
ND
ND
0.005 %
ND
ND
ND
3%

Acknowledgements
This study was funded in part by a Small Business Innovation Research Grant from the National
Institutes of Environmental Health Sciences (ES 08372-03). IVO and LG acknowledge financial
support by the Institute of Veterinary Expertise (Ministry of Public Health) and the Office forScientific, Technological and Cultural Affairs (Prime Minister’s Service).
References
1. Denison, M.S.; Heath-Pagliuso, S. Bul.l Environ. Contam. Toxicol. 1998, 61, 557-568.
2. Garrison, P.M., Tullis, K., Aarts, J.M.M.J.G., Brouwer, A., Giesy, J.P., and Denison, M.S., (1996)
Fundam. Appl. Toxicol. 30, 194-203.
3. Brown, D., Kishimoto, Y., Ikeno, O., Chu, M., Nomura, J., Murakami, T., Murata, H., (2000)
Organohal. Comp. 45, 200-203.

404

ORGANOHALOGEN COMPOUNDSVol. 58 (2002)