ANAM

Application ofthe CALUX {shem'ical activated luciferase gene expression) assay formeasuring TCDD-equivalents in sediment, pore water and blood plasma samples
Murk AJ, A Jonas, A Brouwer
Department of Toxicology, Agricultural University, PO Box 8000, 6700 EA Wageningen, The
Netherlands
PEG Leonards
Institute for Environmental Studies, Free University, De Boelelaan 1115, 1081 HV Amsterdam, The
Netherlands
MS Denison
Department of Environmenlal Toxicology, University of Califomia, Davis, CA 95616, USA

1. Abstract
The results presented in this paper demonstrate that the in vitro C^i(,/A'(cheniical activated luciferase
gene ej;pression) bioassay is a rapid, sensitive assay for assessing the toxic potency of (mixtures ot) Ahreceplor active compounds in sediment, pore waler and blood plasma. Recombinant ral (H4IIE) and mouse(Hepalclc7) hepatoma cell lines, containing arylhydrocarbon receptor (AhR)-medialed luciferase gene
expression, were used to determine the C/lIf/A-response of sample extracts. Using a 2,3,7,8telrachlorodibenzo-p-dioxin (TCDD) standard curve, thc CALUX responses for the samples wereconverted into so-called CALUX-'YEQs (TCDD-equivalents). The detection limit ofthe CALUX assay was
0.5 fmol of TCDD-equivalents. The CALU.Y activity induced by organic extracts from 450 mg aliquotsof sediment or 250^.^1 aliquots of pore-water conesponded well wilh the instramcntally analysed level of
dioxin-like compounds in the sediment. Using pore water, only a simple and rapid extraction procedure
was needed, withoul addilional clean-up lo prevent cell death. The C.4if/A'response bolh for the fatly,uncleaned organic extract and the cleaned extract of blood plasma from experimentally exposed eider
ducks, correlated significantly with the PCB-levcls measured in abdominal fal or blood plasma, and wilh
the hepatic EROD activity in the same animals.
An important advantage ofthe C/IZ, t/A'-assay compared to the ethoxyresomfin O-deethylase (EROD)
assay is that the C/lLf/A''assay is insensitive to substrate inhibition, which is demonstrated in this paperfor 3,3',4,4'.5-pentachlorobiplienyl (PCB-126).
2. Introduction
For hazard and risk assessment of mixtures of PHAHs thc concentrations oflndividual PHAHs multipliedby their respective Joxic equivalency factors (TEFs) are added up to give the total TCDD loxic
equivalency ofthe mixture'. Given the complexity ofthe mixmres of PHAHs in sediments and organisms,
chemical analysis can only give a rough impression ofthe potential health risks. Due to the oflen small
concentrations oflndividual congeners and the presence of unknown or nol routinely measured AhR active
substances, there is a risk of imdereslimation of the lolal TEQ. The toxic responses of AhR active
compounds may be additive, as is the case for polychlorinated dibenzo -p-dioxins (PCDDs) and -furans(PCDl's). but for PCBs and non-PCB like substances both additive and antagonistic interactions have been
observed-'^. These limitations are drawbacks to the TEQ-approach. The C/l/.t/A" bioassay provides a

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measure ofthe loxic potency ofthe whole mixture, including interactions.
This paper presents some examples ofthe use ofthe CALUX assay for monitoring Ah-receptor active
compounds in sediment, pore waler and blood plasma. Applications ofthe CALUX-assay for human milk
and blood samples, butter fat and coconut oil, otter liver and whole blood, and fish samples*'^-*, andapplication ofthe CALUX assay for compoimds that bind to and activate the estrogen receptor', will bepresented separately al Dioxin 1996. In pursue ofthe substrate inhibition tests wilh Clophen A50 and
PCB-77 reported elsewhere^, possible substrate inhibition ofthe more potent Ah-receplor agonist PCB126 is also determined, by comparing the dose-response curves in the EROD and C.4LUXassay.
3. Methods
Sample collection and preparation
Sediment samples differing in degree of pollution were obtained from the National Institute for Inland
Water Management (RIZA)were decanted and sieved, ten grams of sediment was mixed with Na2S04,
dried in an oven ovemight al 40°C, and extracted with hexane:acetone (1:1) in a Soxhlet for 16 hr*.
Sulphur was removed using tetra butyl ammonium sulphite (TBA). Further clean-up was performed using
a multi-layer acid-base silica column consisting of 0.75 g Na2S04, on top of dried silica with 0.75 g of
22% and 0.75 g of 40% hexane washed H2SO4, and I g of 33% NaOH on glass wool. The column was
eluted with 20 ml of hexane followed by 20 ml of hexane:dichloromethane (1:1), dried under a gentle,
filtered air-flow, and dissolved in 100 |il of DMSO. Pore waler was collected from 200 g decanted andsieved sediment by cenlrifugation for 30 min al 3000 g al 5°C. The supernatant was carefully decanted
into glass erienmeyer flasks and stored at 5°C. Samples of 5 ml pore waler were extracted three times with
5 ml hexane, the hexane was evaporated under a gentle nitrogen flow, and thc extract dissolved in 200 plof DMSO. Blood plasma was collected from 27 days old eider ducklings, was collected on day 10 after
i.p. injection wilh 5 ml com oil/kg body weight (bw), 5 or 50 mg PCB-77/kg bw, or with 50 or 200 mg
Clophen A50/ kg bw"*. EROD activities were measured in hepatic microsomes, and PCB levels in
abdominal fat (using GC-ECD)''. Blood plasma aliquots of aboul 1.5 ml were denaturated with an equalamounl of methanol. The PHAHs were extracted three times wilh 3 ml of hexane. After the first extraction
step, 3 drops of 6 M HCI were added lo the waler phase. Part ofthe extract was laken for fiu'ther clean-up,
the rest was evaporated at 30 °C under a gentle sU'eam of nitrogen gas, and dissolved in DMSO for use in
the CALUX assay using Hepa.Luc cells. For the firsl dilution step these fatty exU'acts had to be kept warm(±30°C) to prevent clotting ofthe lipids in the pipcl. TTie other part ofthe extact was cleaned over a 20%
H2SO4 deactivated silica column with hexane-diethyl ether (97:3, v/v). This extract was evaporated,dissolved into isooctane, and analysed by GC. After GC-analysis, the remaining extract was evaporated
and dissolved in DMSO for measurement of C/^it/A-activity in H4IIE.Luc cells.
Forthe CALUX-assay, H411E.pGudlucl.l (H41IE.Luc) or Hepal clc7.pGudluc (Hepa.Luc) cells prepared
as previously described^'' were exposed to PHAHs in 24-well culture plates*. Cells were seeded in 500
pl growth medium and incubated for 24 hr until the cell layer was 80-90% confluent. The niediimi was
replaced by fresh growth medium containing the tesl compound in maximal 0.5% DMSO. After 24 hours
incubation the cells were rinsed twice wilh 50% diluted PBS (0.5* PBS), 75 pl lysis mix was added, thecells were harvested 15 minutes later and centrifuged for 90 sec. at 13000 g. The supematant was frozen
at -80°C. For luciferase measurement 20 pl supematant was pipetted in a 96-well microtiter plate, 100 nl
luciferin assay mix (Promega) was added and after 90 sec mixing on a plate mixer the light production wasmeasured in an Amerlite Luminometer. For calculation of C/fif/A-TEQs a standard curve of TCDD was
filled, and the CALUX-TEQ value for the unknown sample was interpolated on this curve*. For thesubstrate-inhibition lest the CALUX assay has been performed in 96-well culture plales. Briefly,
H4IlE.Luc cells vvere seeded in 96-well plales in 100 pl growlh medium. After 24 hr incubation at 37 °C
the cell layer was 80-90% confluent, and 100 pl of fresh medium containing the test compound in
maximal 0.5%) DMSO was added lo each well. After an addilional 24 hr incubation, the plales were rinsed

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twice with 0.5*PBS, 20 pl lysis buffer was added, and the plates were placed at -80°C. The plates were
measured in a Labsystems Luminometer (Merlin) with automated injection. To bc able lo correct fordifferences in quantification due to assay to assay variation, three TCDD calibration standards were
measured wilh each assay.
llic EROD assay was mainly based on the melhod described before'". Briefly, H4IIE wild lype cells wereseeded in 96-vvell plales and exposed as described for the CALUX assay in 96 well plates, but exposure
was during 48 hr instead of 24 hr. Instead of lysis mix, 20 pl of nanopurc water was added before theplales were placed al -80°C. To measure rcsomfin production, the plates were pre-incubatcd for 20 min.
al 37°C, wilh 50 pl of Tris-sucrose buffer (pH 8) wilh 40 pM dicumerol, and 25 pl 10 pM 7elhoxyresomfin (ER) was added lo each well. The reaction was started with 25 pl 1 mM NADPH per well,and after I hr incubation at 37°C, the resomfin production was measured in a fluorometer (Cytofluor) wilh
excitation at 530 nm and emission at 590 nm.
For the CALUX or EROD Substrate inhibition assay, H41IE.Luc or H41IE wild type cells were exposed
to 50 pM TCDD in 96-well plates for 24 hr, and frozen at -80°C as described above. The plales were
thawed on ice prior to luciferase or EROD activity measurement, and final concentralions of PCB-126
ranging from 1 to 10000 nM were reached by adding appropriate concentrations of slock solutions in 4
pl DMSO to each well. The plates were thoroughly mixed on a plale mixer before CALUXoi EROD
activity were measured as described above.
GC-ITD analysis was perfomied using the isooctane extract of eider duck blood plasma (sec above). ITiegas chromatography (GC) column DB5-MS, 30 m x 0.2 ^m (J&W Scientific) was coupled to a Saturn II
ion trap detector (ITD; Varian). A retention gap of 2 m x 0.53 mm i.d. deactivated fused silica was used
(Chrompack). ITic GC and ITD conditions are described elsewhere'-'.
4. Results and discussion
The C.4/.L'A'response was found to be very sensitive and reproducible using PCDD as a positive conlrol.
The detection limil in H41IE.Luc cells was less than 1 pM, which equals an absolute amount of less than
0.5-0.2 fmol/well, depending on method of exposure used*. The EC5Q was 10 pM, the TCDD doseresponse curve saturated belween 100 pM and 1 nM, and the slandard deviation was generally < 5%.
Exposure of H4IIE.Luc cells to extracts of Ketelmeer (KM) sediment, which is known lo be polluted
with several PHAHs, resulted in 17 fold grealer C/IZ.LC^'activity than did extracts fi'om die relatively clean
Ooslvaardersplassen (OVP) sediment*. Based on chemical analyses, the TEQs of these two locations

15,

c
o

0' 0.11

10100^1 pore water

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+

OVP

o

KM

A

NSC

Figure 1 The CALUX response
by pore water extracts in H411E.Luc cells, expressed as induction
factor relative to backgroundinduction. OVP= Ooslvaardersplassen, NSC= North Sea Canal,
KM= Ketelmeer

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differed by a factor 20. A comparable difference was observed using pore waler extracts. Pore water fromthe North Sea Canal (NSC) conlained almost 13 fold more CALUX-TEQs than the polluted KM (Figure1). The high signal in the NSC was probably caused by accidental emissions of PCDD and PCDF froma herbicide producing plant situated in the NSC. Analysis of only the solid phase of sediments does nol
discriminate between bioavaiiable and tightly bound contaminants, where pore water samples represeni
the biologically available fraction of sediment contamination. An advantage of analysis of pore water isthat no soxhlet extraction is needed, or clean-up to prevent cell-death. This makes the sample preparationmuch more rapid and the chance of loosing unknown AhR-active compounds with yet unknown optimal
recovery conditions, much smaller.

3

10000

o

1000

-•V
100

o

1 /

10

UJ
X
3

<
o

y-0.94x+0.15

1

1

r-0.97

Figure 2 Correlation between
CALUX TEQ of blood plasma
extracts from experimentally
exposed eider ducks, with andwithout clean-up.

10
too
1000
10000
CAtUX TEQ fatty extracts (fmol/ml)

Extracts of eider duck blood plasma's induced C/fZ. (/A'responses in a dose-related manner (Murk el al.,submitted). The CALUX-TEQs based on the fatly blood plasma extracts conelaled significantly with either
the PCB-levels in die abdominal fat (r= 0.80-0.94, depending on die PCB-congener used), widi die hepatic
EROD activity of die eider ducks (i=0.88), and with die CALUX-TEQs determined for die cleaned exU'acts(Figure 2; r=0.96). Also die PCB levels in blood plasma extracts correlated well with the CALUX-TEQs
measured in these cleaned extracts (r=0.86-0.96, depending on the PCB-congener used) (data not shown;
Murk et al., submitted). These results demonstrate that blood plasma extracts can be measured in the
CALUX assay wilh and without a clean-up procedure. Performing a simple Silica-H2S04 clean-up,
however, offers the possibility to concenU'ale the exU'act, and makes it easier to manipulate the extract. The
observed good conelation ofthe CALUX-TEQ in blood plasma with the PCB-levels in abdominal fal
suggests that the C.4LU.Y-TEQ in blood plasma provides an integrated measure of toxic potency ofthe
inlemal dose ofthe AhR active PHAHs. Allhough the experimentally dosed eider ducks used for this study
contained relatively high levels of PCBs, die CALUX-assay has already been applied for measuring TEQs
in environmentally exposed species, such as cormorants, otters* and himians'*. About 0.5-1 ml of bloodplasma is needed for quantifying CALUX-TEQs in blood plasma, depending on the (expected) level of
exposure (Murk et al., submitted).
Substrate inhibition
The CALUX sssay oflers some advantages when compared to the commonly used EROD assay in H4IIE
cells, ll is slightly more sensitive and has a three fold greater induction factor*''^, but, more importantly,
die use ofthe C/lZ.t/A'assay is not limited by substrate inhibition*. As is shown in Figure 3, the CALUX
and EROD induction both increase with increasing PCB-126 concentration. The EROD activity, however,decreases again at concentrations greater than 100 nM, while the C/tZ-C/A'activity saturates at the
maximum level (Figure 3). For the potent Ah-receptor agonist PCB-126, maximimi induction levels are

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not significantly differeni from the maximum induction wilh TCDD. However, the maximum EROD
induction declines with decreasing AhR inducing potencies of PHAHs (Murk el al., in prep.). Figure 4
demonstrates the CALUX and EROD activity, induced by 50 pM of TCDD, when increasing amounts of
PCB-126 are added to the lysed cells shortly before adding the respectively subsfrates. The measured
EROD activity was already reduced to 73% ofthe original activity by addition of 10 pM of PCB-126, andafter addition of 10 nM PCB-126 only 6% ofthe EROD activity is left. No inhibition ofthe CALUX
activity was observed al any ofthe PCB-126 concenlralions lested, which was to be expected, since
PHAHs are not substrates for luciferase. Thesefindingare in accordance with previously reported results*
for H4IIE cell lines, and with resulls wilh experimentally dosedflounder,where the EROD activity
induced by 5 pg TCDD/kg was strongly reduced when dosed simultaneously with Clophen A50/kg,
although the cytochrome P450 protein content increased in an additive manner'''.

EROD(PCB126)
EROD(TCDD)CALUX(PCB12e)
CALUX(TCDD)

i/y/Figure 3 CALUX and EROD
induction by PCB-126 in resp.
H4IIE.LUC and H4IIE.wt cells.
Exposure was during 24 hr in 96well culture plates. Maximum
induction was reached by 1000 pM
of TCDD.

Compound (pM)

140
Q

Q
O
1-

120-

•F

100 <t —

OL

O
IO

—^^^

80-

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^
at
CO

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at
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60 J
4020-

0
10

100

1000

10000

50000

PCB-126 (nM)

Figure 4 Effect of an increasing
concentration of PCB-126 added tolysed H4IIE.LUC or H4IIE.wrt cells
shortly before measuring the
CALUX or EROD activities, whichwere induced by exposure to 50 pM
TCDD during 24 hr (this induction
was set at 100%).

5. Acknowledgements
Tliis study was financially supported by the Dutch Technology Foundation (STW), grant nr. WB122.2823.
J Legler, A Bulder and M Rozemeijer contributed to parts ofthe work described in this short paper.

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6. References' Ahlborg UG, GC Becking, LS Bimbaum, A Brouwer, HJGM Derks, M Feeley, G Golor, A Hanberg,
JC Larsen, AKD Liem, SH Safe, C Schlatter, F Waem, M Younes, and E Yrjanheikki (1994): Toxic
equivalency factors for dioxin-like PCBs-Report on a WHO-ECEH and IPCS consultation, December
1993. Chemosphere 28:1049-1067.
^ Safe SH (1994): Polychlorinated biphenyls (PCBs): environmental impact, biochemical and toxic
responses, and implications for risk assessment. Cril. Revs.Toxicol. 24, 87-149.
^ Aarts, JMMJG, MS Denison, MA Cox, AC Schalk, PA Ganison, K Tullis, LHJ de Haan and A Brouwer.
(1995): Species-specific antagonism of Ah receptor action by 2,2',5,5'-telrachloro- and 2,2'3,3'4,4'hexachlorobiphenyl. Eur.J.Pharm.Env.Tox.Section 293,463-474.
•* Aarts JMMJG, PH Cenijn, BMG Blankvoort, AJ Murk, A Brouwer, TFH Bovee, WA Traag, LAP
Hoogenboom, S Patandin, N Weisglas-Kupems, PJJ Sauer and MS Denison (1996): Application ofthe
chemical-activated luciferase expression (CALUX) bioassay for quantification of dioxin-like compounds
in small samples of human milk and blood plasma.. Short paper Dioxin 1996 Amsterdam.
' Bovee, TFH, LAP Hoogenboom, WA Traag, T Zuiderman, JHJ Horstman, JMMJG Aarts, AJ Murk and
HA Kuiper (1996): Biological screening of Ah receptor agonist activity in butter fal and coconut oil bymeans of chemical-activated luciferase expression in a genetically enginered cell line (CALUX). Shortpaper Dioxin 1996 Amsterdam.
' Murk AJ, PEG Leonards, R Luit, B Spenkelink, B van Hattimi, M Smil. Monitoring exposure and effecisof polyhalogenated aromatic hydrocarbons (PHAHs) in European otters (Lutra lutra). Short paper Dioxin
1996 Amsterdam.
^ Legler J, LM Bou-wman, AJ Murk, A Brouwer, J Stronkhorst and D Vethaak (1996): Determination ofdioxin- and estrogen-like activity in sediment extracts using in vitro CALUX assays. Short paper Dioxin
1996 Amsterdam.
* Murk, AJ, J Legler, MS Denison, JP Giesy, C van de Guchte and A Brouwer.Chemical activatedluciferase expression (CALUX): a novel in vitro bioassay for Ah receptor active compounds in sediments
and pore waler. Subm. Fund. Appl. Toxicol.
' Garrison, PM, K Tullis, JMMJG Aarts, A Brouwer, JP Giesy and MS Denison (1996): Species-specificrecombinant cell lines as bioassay systems for the detection of 2,3,7,8-tetrachlorodibenzo-p-dioxin-likechemicals. Fundamental and Applied Toxicology. 30, 194-203.
'" Murk AJ, JHJ Van den Berg, M Fellinger, MJC Rozemeijer, C Swenncn, P Duiven, JP Boon, A
Brouwer and JH Koeman (1994): Toxic and biochemical effects of 3,3',4,4'-letrachlorobiphcnyl (CB77)and clophen A50 on eider ducklings (Somateria mollissima) in semi-field experiment. Environ. Poll. 86,
21-30.
" Rozemeijer MJC, JP Boon, C Swcnnen, A Brouwer and AJ Murk (1995): Dioxin type and mixed lype
induction of the cytochrome P450 syslem of common eider (Somateria mollissima) by PCBs: withindications for biotransformation. Aqual. toxicol. 32, 93-113.
'^ Sanderson, JT, JMMJG Aarts, A Brouwer, KL Froese, MS Denison and JP Giesy (1996): Comparisonof receptor-mediated Luciferase and Ethoxyresomfin-0-deethylase induction in H4IIE cells: Implications
for their use as bioanalytical tools for the detection of polyhalogenated aromatic hydrocarbons. Toxicology
and Applied Pharmacology. 137, 316-325.
'•* Leonards, PEG, UA Zierikzee, Th Brinkman, WP Cofino, NM van Straalen and B van Hattum
Submitted: Exposure of otters (Lutra lutra) lo planar PCBs accumulated in an aquatic food web. Submitted
lo Environ. Toxicol. Chem.
'^ Besselink HT, AD Vethaak and A Brouwer (1996): Effect on refinoid homeostasis in the flounder(Platichthys flesus) following chronic exposure to contaminated harbour dredge spoil from Rotterdam
harbour. Short paper Dioxin '96, Amsterdam.

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