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Author Manuscript
Talanta. Author manuscript; available in PMC 2012 December 17.
Published in final edited form as:
Talanta. 2011 October 15; 85(5): 2484–2491. doi:10.1016/j.talanta.2011.07.103.

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Quantification of PCDD/Fs and dioxin-like PCBs in small
amounts of human serum using the sensitive H1L7.5c1 mouse
hepatoma cell line: optimization and analysis of human serum
samples from adolescents of the Flemish human biomonitoring
program FLEHS II
K. Croes*,1,8, K. Van Langenhove1, E. Den Hond2, L. Bruckers3, A. Colles2, G. Koppen2, I.
Loots4, V. Nelen5, G. Schoeters2, T. Nawrot6,7, N. Van Larebeke8, M.S. Denison9, T.
Vandermarken1, M. Elskens1, and W. Baeyens1
1Vrije Universiteit Brussel, Department of Analytical and Environmental Chemistry (ANCH),
Pleinlaan 2, 1050 Brussels, Belgium
2Flemish Institute for Technological Research (Vito), Environmental Health and Risk, Boeretang

200, 2400 Mol, Belgium$watermark-text

3Interuniversity Institute for Biostatistics and Statistical Bioinformatics, Hasselt University, Belgium
4University of Antwerp, Political and Social Sciences, Antwerp, Belgium
5Provincial Institute for Hygiene, Antwerp, Belgium
6Occupational & Environmental Medicine, Unit of Lung Toxicology, K.U.Leuven, Belgium
7Centre for Environmental Sciences, Hasselt University, Diepenbeek Belgium
8Ghent University Hospital, Study Centre for Carcinogenesis and Primary Prevention of Cancer,

Ghent, Belgium
9Department of Environmental Toxicology, University of California, Davis, CA 95616, USA$watermark-text

Abstract
Since the CALUX (Chemically Activated LUciferase gene eXpression) bioassay is a fast andinexpensive tool for the throughput analysis of dioxin-like compounds in a large number ofsamples and requires only small sample volumes, the use of this technique in humanbiomonitoring programs provides a good alternative to GC-HRMS. In this study, a method for the
separate analysis of PCDD/Fs and dioxin-like PCBs (dl-PCBs) in human serum with the newsensitive H1L7.5c1 mouse hepatoma cell line was optimized.
Sample dilution factors of 5 and 2.4 were selected for routine analysis of respectively the PCDD/
Fs and dl-PCBs. The validation studies showed that repeatability and within-lab reproducibility for
the QC standard were within the in-house criteria. A long-term within-lab reproducibility of 25%
for the PCDD/F fraction and 41% for the dl-PCB fraction for the analysis of pooled serum
samples, expressed as pg BEQ/g fat, was determined. CALUX recoveries of the spiked proceduralblanks were within the acceptable in-house limits of 80–120% for both fractions and the LOQ was
30.3 pg BEQ/g fat for the PCDD/Fs and 14.5 pg BEQ/g fat for the dl-PCBs. The GC-HRMS
recovery of a C13-spiked pooled serum sample was between 60–90 % for all PCDD/F congeners
and between 67–82 % for the non-ortho PCBs. An adequate separation between both fractions was*corresponding author Kim Croes, Tel: +32 (0)2 629 32 93, Fax: +32 (0)2 629 32 74, kim.croes@vub.ac.be.

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found. The CALUX/GC-HRMS ratio for a pooled serum sample was respectively 2.0 and 1.4 for
the PCDD/Fs and the dl-PCBs, indicating the presence of additional AhR active compounds. As
expected, a correlation was found between human serum samples analyzed with both the new
H1L7.5c1 cell line and the more established H1L6.1c3 cell line. The geometric mean CALUXBEQ values, reported for the adolescents of the second Flemish Environment and Health Study(FLEHS II) recruited in 2009–2010, were 108 (95% CI: 101–114) pg CALUX-BEQ/g fat for the
PCDD/Fs and 32.1 (30.1–34.2) pg CALUX-BEQ/g fat for the dioxin-like PCBs.

Keywords$watermark-text

PCDD/Fs; dioxin-like PCBs; CALUX; human serum; biomonitoring; FLEHS II

1. Introduction
Although emissions of PCDD/Fs and PCBs have decreased during recent years, thesecompounds are still environmental pollutants of concern: 1) since PCDD/Fs and dioxin-like
PCBs are persistent in the environment, accumulate in fat tissue and in the food chain, havehormone disrupting properties and are carcinogenic [1, 2] and 2) because emissions incertain locations in Flanders are still high [3]. Therefore, it is important to include theanalysis of these compounds as exposure biomarkers in human biomonitoring programs.

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In 2007, a second cycle of the Flemish Human Biomonitoring program (FLEHS II) started
and more than 40 biomarkers of exposure (i.e. metals, persistent organic pollutants,
perfluorinated compounds, …) and 10 effect markers (i.e. hormones) were measured in 650
samples, recruited from 14–15 year-old adolescents (n=200), adults between 20–40 years(n=200) and mother-child pairs (n=250) [4]. Since only a small amount of serum (5 mL) was
available for the PCDD/F and dioxin-like PCB determination, screening of these samples by
GC-HRMS analysis was not possible, since the individual congeners would be below thequantification limit when using such low sample volumes. The CALUX bioassay provided agood alternative, since it requires only a small amount of serum to analyse the total amountof dioxin-like compounds in the extract.

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This publication presents an optimized method for the separate analysis of PCDD/Fs anddioxin-like PCBs in human serum with the newly developed and more sensitive third
generation CALUX (H1L7.5c1) mouse hepatoma cell line [5, 6]. The H1L7.5c1 cell line
was specially designed to analyze low concentrations of PCDD/Fs and PCBs in small
sample volumes. With the less sensitive H1L6.1 cell line, which was commonly used inprevious biomonitoring studies [7, 8, 9] and food/feed analysis [10, 11], only a single-point
analysis of the whole extract was often used and it was not possible to measure the dioxinlike PCB fraction, since most samples were below the quantification limit (LOQ) [9]. In thisstudy, for the first time, dioxin-like PCBs could be measured in serum samples with the
improved H1L7.5c1 cell line with a high percentage of the samples above the LOQ. Doseresponse analysis using different dilutions of serum sample extracts allowed determinationof an optimal dilution factor to facilitate screening analysis and to minimize sample volumes
needed for analysis. The use of this new H1L7.5c1 cell line will also allow optimization of
CALUX protocols for the analysis of both PCDD/Fs and dioxin-like PCBs in various
matrices, especially those with low concentrations and/or small sample volumes like foodand feed or human samples (i.e. blood and milk).

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2. Materials and Methods
2.1 Chemicals and standards$watermark-text

Hexane (for dioxins and PCBs, minimum 96%), acetone (Pesti-S grade, minimum 99.9%)
and toluene (for dioxins and PCBs, minimum 99.8%) were purchased from Biosolve (TheNetherlands). Ethyl acetate pestanal and silica gel 60 for column chromatography werepurchased from Sigma-Aldrich (Germany). Sulphuric acid (95%–97%, ACS reagent), Celite
545 (0.02–0.1 mm) and DMSO were obtained from Merck (Germany). Anhydrous sodium
sulphate was purchased from Boom (The Netherlands) and the X-CARB from XDS (USA).
The standard solution of 2,3,7,8 TCDD (50 ng/mL) was purchased from Campro Scientific(The Netherlands).
2.2 Analytical procedure$watermark-text

Extraction and clean up—The extraction and clean up procedure for the analysis of
PCDD/Fs and dioxin-like PCBs was based on the protocol used by Schroijen et al. (2006)
and Van Wouwe et al. (2004) [12, 13] with some small adaptations. Briefly, 5 mL of humanserum was weighted and mixed with 15 mL of acetone for 2 min in order to denature theproteins. PCDD/Fs, PCBs and other lipophilic compounds were extracted 3 times with eachtime 5 mL of hexane using shaking for 2 minutes, followed by centrifugation for 2 minutes.
This hexane solvent phase was filtered upon a pre-conditioned celite column, filled with 0.5
g (1.3 cc) of celite and 6.5 g (4.3 cc) of anhydrous sodium sulphate and conditioned with 30
mL of hexane. The hexane fractions containing serum lipids were collected in a glass tube
and the celite column was then washed with 10 mL of hexane, which was also collected in
the tube, to elute all lipids from the column. After extraction, the samples were gently
evaporated at 40 °C under a flow of pure air until only the serum lipids remained. The
amount of fat was weighted and the extract was redissolved in 5 mL hexane and cleaned up
on a pre-conditioned multi-layer silica column coupled in series with a carbon column.

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The silica gel column (25 mL) was filled from bottom to top with glass wool, 1.9 g (1.3 cc)
sodium sulphate, 3.0 g (4.3 cc) of 33% (w/w) sulphuric acid silica gel and 1.9 g (1.3 cc)
sodium sulphate. The carbon column (10 mL) was filled with glass wool, 0.7 g (0.5 cc)
sodium sulphate, 0.34 g (1 cc) X-CARB and 0.7 g (0.5 cc) sodium sulphate. Before loading
the sample extract, the silica column was rinsed with 30 mL hexane, while the carbon
column was conditioned with respectively 5 mL acetone, 20 mL toluene, 10 mL hexane.
After sample addition, the glass tube containing the serum lipids was rinsed with 2 times 5
mL hexane and finally the column was eluted with 15 mL hexane. After full elution, theupper acidic silica column was removed and the carbon column was eluted with 8 mL of ahexane/acetone (90/10) mixture. Because this fraction was toxic to the cells, it was discarded[14]. The coplanar PCBs were eluted from the carbon column with 15 mL of hexane/ethyl
acetate/toluene (80/10/10), followed by elution of the PCDD/Fs with 20 mL toluene. The
PCDD/F and PCB fractions were evaporated until dry (40 °C) and redissolved in a definedvolume hexane. The samples were stored at room temperature until CALUX analysis.
CALUX bioassay—The CALUX (Chemical Activated LUciferase gene eXpression) assay
is a reporter gene mammalian cell bioassay. The recombinant cells used in the bioassay
contain a stably transfected AhR-responsive firefly luciferase reporter gene, which respondsby the induction of luciferase. The cell lines used in the bioassay were the recombinantmouse hepatoma cell lines H1L6.1c3, stably transfected with pGudLuc 6.1 containing 1
dioxin responsive domain (1 DRD), and the more sensitive H1L7.5c1 mouse hepatoma cellline, stably transfected with pGudLuc 7.5 and containing 5 dioxin responsive domains (5DRDs) [5, 6]. Cell treatment and measurement were based on the protocols described byWindal et al. (2005) [14] and the XDS method 4435 from 2008. Briefly, the cells were

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grown in cell culture plates containing 15 mL RPMI 1640 supplemented with 8% FCS and
1% penicillin/streptomycin (Gibco, UK). After trypsinization, the cells were counted anddiluted to a concentration of 55 - 65E4 cells per mL and each well of the 96-well plate wasseeded with a 200 µL cell suspension in RPMI. After 24 h of incubation (37 °C, 5% CO2),
the 96-well plate was seeded with the standard solutions and sample extracts. Therefore, thetarget compounds in the sample extracts were transferred to 4 µL DMSO by evaporation ofhexane at 40 °C using a vacuum centrifuge. Then, 400µL RPMI medium was added and the
mixture was dosed to the cell lines in duplicate (2 wells, 188µl per well). When 188 µL of astandard solution, blank or sample extract in RPMI with 1% DMSO was added to every
well, the plate was again incubated for 20 – 24 h. Afterwards, the medium was removed, thewells were rinsed with 75 µL PBS buffer pH 7.4 (Gibco, UK) and the cells in each well werevisually inspected under the microscope in order to evaluate if the extract was toxic to thecells. If not, 50 µL lysis reagent (Promega, USA) was added to each well and the plate wasshaken for 5 minutes. After a 10 minute incubation period in the luminometer (Glomax,
Promega, USA), 50 µL luciferin reagent (Promega, USA) was injected and the light output
was given in Relative Light Units (RLUs) (integration time 5 s for the H1L6.1c3 and 3 s for
the H1L7.5c1, lag time of 5.6 s for both cell lines). A four parameter Hill-function was used
to fit a sigmoid curve through the standard solutions. The measured luminescence in relative
light units (RLU) of an unknown sample was converted into a bioassay toxic equivalency
value (CALUX-BEQ) by comparison of the response of the sample to the 10-point sigmoid
dose-response curve obtained with 2,3,7,8-TCDD standards (concentration range of 50000–
97.7 fg/well for H1L7.5c1 and 12500–24.4 fg/well for the H1L6.1c3). Three quality control(QC) solutions (i.e. a standard solution of TCDD corresponding to a RLU induction ofaround 50%) and 3 DMSO blanks were added in duplicate to every 96-well plate as aninternal control. The PCB fractions were measured with the sensitive H1L7.5c1 cell line,
since the concentrations were very low. The PCDD/F fractions could be measured using
either the H1L6.1c3 or the H1L7.5c1 cell line. However, the % RLU induction is very low
with the H1L6.1c3 cell line. And therefore the H1L7.5c1 cell line, which gave % RLU
inductions near the EC50 value for the pooled samples, is preferably used for both theanalysis of PCDD/Fs and dl-PCBs in small volumes of human serum samples.

3. Optimization and validation parameters
3.1 Quantification limit and EC50 calculation$watermark-text

A detection limit (LOD) shows that the signal is significantly higher than the blank(background value). To calculate the concentration of chemical in a sample, the signal
produced by that concentration needs to be quantifiable. The limit of quantification (LOQ) is
defined as the compound concentration which gives a luminescent signal that is equal to the
DMSO blank plus 6 times the SD of that mean DMSO blank signal (6 wells/ plate).
However, when using a sigmoid calibration curve, the LOQ is normally defined as the lower
limit of the working range (indicated by the arrows in Figure 1). Using this definition, theLOQs for the H1L6.1c3 and H1L7.5c1 cell lines were 0.30 and 0.10 pg CALUX-BEQ/well,respectively. Occasionally, in case of high DMSO concentrations and/or high variability
between the wells (high SD), the calculated LOQ (DMSO+6*SD) could be higher than thehere above defined LOQ of 0.30/0.10 pg CALUX-BEQ/well. Serum samples below thecalculated LOQ values should in this case be reanalyzed with the CALUX assay for anadequate BEQ-quantification. For the more sensitive H1L7.5c1, the LOQ of 0.10 pg
CALUX-BEQ/well corresponded to 14.5 pg CALUX-BEQ/ g fat for the PCBs and 30.3 pg
CALUX-BEQ/ g fat for the PCDD/Fs, taking into account a mean amount of fat in theserum sample (5 mL) of 0.0165 g for adolescents and a total dilution factor of 2.4 for the dlPCBs and 5 for the PCDD/Fs. This means that the H1L7.5c1 cell line was three times moresensitive than the H1L6.1c3 cell line. At the EC50 value, which was defined as theconcentration corresponding to the minimum induction level plus half of the difference
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between the maximum and minimum induction levels (% RLU of the), the H1L7.5c1 cell line was even four times moresensitive than the H1L6.1c3 cell line (mean EC50 values of 438.6 and 1804.5 fg/wellrespectively).
3.2 Full dose-response curves of dioxin-like compounds in human serum samples
When analyzing serum samples, it is important to determine the optimum dilution factor.
The most efficient way to do this is by analysis of different sample dilutions from the same
sample or the same pool of samples.

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In this study, dilutions curves were obtained for pooled human serum samples from 300
adults (50–65 years old). Dilutions of 1.2, 1.5, 2, 4, 5, 8, 31 and 40 for the PCDD/F fraction
and dilutions of 1.2, 1.5, 1.67, 2.4 and 4 for the dl-PCB fraction, obtained from the samepooled serum sample, were analyzed with the H1L7.5c1 cell line.
Figure 2 shows two full dose-response dilution curves for the PCDD/F fraction, measured
with the H1L7.5c1 cell line. The samples were from the same pool, but extraction, clean up
and bioassay measurements were done independently for both curves. Table 1 gives an
overview of the CALUX-BEQ in pg/g fat for the PCDD/F fraction for the different dilution
points from the full dose curves in Figure 2. The CALUX-BEQ was calculated for eachdilution factor using the solver function on the TCDD standard calibration curve and taking
into account the amount of fat and the used dilution factor.

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From Figure 2 and Table 1 it is clear that the optimum dilution factor (df) is in the range of4, 5 and 8, with induction levels between 70 and 49 % and BEQ-values ranging from 66.2 to102.3 pg CALUX-BEQ/g fat. When incubating cells with more diluted samples (df 20 to40), the induction levels were rather low, which resulted in an overestimation of the
CALUX-BEQ. Also, serum samples with low PCDD/F levels would be below thequantification limit (LOQ). When dosing more concentrated samples (df 1.2, 1.5 or 2), the% RLU induction was again lower than the curve maximum (past the top of the curve) andthe sample extracts were in the so called “toxic range” of the calibration curve. This resulted
in very low CALUX-BEQ values (Table 1) and thus an underestimation of the PCDD/F
concentration. Since no cell death was observed in these cells, there were probablyinterfering compounds from the serum matrix that suppressed the CALUX signal and/or the
induction response. More research is needed to identify these interfering compounds.

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Measurements of the PCDD/F fraction with the H1L6.1c3 cell line gave similar doseresponse curves with the same curvature, but the maximum induction level was only 12.5 %(compared to 70 % with the H1L7.5c1 cell line) at df 4. For the PCB fraction, dilution
factors 1.2, 1.67, 2.4, 3.3, 4 and 5 were tested. The optimal dilution factors for the H1L7.5c1
cell line were between 1.2 and 2.4; dilution factors 3.3, 4 and 5 yielded responses below thequantification limit. In contrast to the PCDD/F fraction, no “toxic effects” were observed for
the PCB fraction. When using the less sensitive H1L6.1c3 cell line, most PCB extracts were
below quantification limits (data not shown). From this experiment, it was decided to use themore sensitive H1L7.5c1 cell line with a dilution factor of 5 for the PCDD/F fraction (0.8mL out of 2 mL hexane extract, dosed in duplicate) and a dilution factor of 2.4 for the PCB
fraction (1 mL out of 1.2 mL hexane extract, also dosed in duplicate).
3.3 Repeatability and within-lab reproducibility
Repeatability and within-lab reproducibility of the luciferase induction of the cell bioassay
were calculated by analysis of the QC TCDD-standard on different days. On every 96-wellplate, the QC standard solution was analyzed 3 times in duplicate (6 wells/plate) and theTalanta. Author manuscript; available in PMC 2012 December 17.

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Page 6concentration (0.25 pg/well or 125 fg/µl) was chosen such that it would produce an
induction response near the EC50 value (i.e. in the middle of the working range).
Table 2 gives an overview of the repeatability of the QC standard on a 96-well plate (SDrand RSDr), the within-lab reproducibility for analysis on different days (SDRW and RSDRW)
and the CALUX recovery.
From Table 2 it is clear that all parameters of the bioassay analysis were within the quality
criteria in our lab: RSDr and RSDRW were lower than 15% and the recovery was between
80–120%.

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The long-term within-lab reproducibility of the whole procedure (extraction, clean up andbio-assay analysis) was calculated for pooled human serum samples from adults, analyzed
on different days (between September 2009 and February 2011) with the H1L7.5c1 cell line.
Table 3 gives an overview of the results for the PCB and the PCDD/F fractions, expressed in
pg BEQ/g fat and in pg BEQ/g serum. For the PCB fraction a dilution factor of 2.4 wasused, while for the PCDD/Fs a dilution factor of 5 was analyzed. The mean CALUX-BEQ
values were 20.0 pg BEQ/g fat for the dl-PCB fraction and 83.3 pg BEQ/g fat for the PCDD/
F fraction. As expected, the % RSD was higher for the samples than for the QC standards,
since for the serum samples also extraction and clean up contribute to the variability.
3.4 Procedural blanks$watermark-text

To assess the specificity and to account for interferences that may derive from the solventsused and sample treatment methods, procedural blanks were analyzed during every run.
Therefore, 5 mL ultra pure water was extracted, cleaned up and analyzed with the CALUX
assay according to the previously described protocol. To detect possible antagonistic andsynergetic effects, the blank extract from both the PCB and PCDD/F fraction was spiked
with the QC TCDD-standard (section 3.3). The recovery of the spiked blank was comparedwith the recovery of the 3 QC standards on the CALUX 96-well plate and should be
between 80–120 % [15]. For the PCB fraction, the mean recovery from 9 spiked proceduralblanks was 114 % (RSD 5.1 %), while for the PCDD/F fraction (n = 12) a mean recovery of
111 % (RSD 9.1 %) was found. In both fractions, a slightly elevated recovery was found,which could be due to impurities originating from the used solvents or column fillings. In
later experiments, new batches of solvents, silica and XCARB were used and no elevationsof the recovery were found. During all experiments, no cell death was observed in thesamples and the procedural blanks.

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3.5 Recovery and correlation with GC-HRMS
The recovery was calculated from a C13 spiked pooled serum sample, that was measured
with GC-HRMS (VITO, Unit Environmental Analysis and Technology, Mol, Belgium).
Therefore, 8 serum samples (5 mL) from the same sample pool were extracted and cleaned
up with separation of the PCDD/F and dl-PCB fraction, according to the normally used
CALUX protocol, and then pooled together for analysis by GC-HRMS. The serum pool was
the same as the one used to analyze the long-term within-lab reproducibility of the CALUX
assay (Table 3).
Table 4 provides an overview of the CALUX and GC-HRMS results for both the PCDD/Fs
and the dioxin-like PCBs in the sample extracts. For the GC-HRMS analysis, the WHO-TEF
values of 2005 [16] were used for both PCDD/Fs and non-ortho PCBs. The mono-ortho
PCBs were not analyzed by GC-HRMS.
The CALUX-BEQs were higher than the GC-HRMS results. For the PCDD/F fraction a
CALUX/GC-HRMS ratio of 2.0 was found, while for the PCB fraction the CALUX
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response was 40% higher compared to the GC-HRMS TEQ-values. Most authors reportCALUX PCDD/F fraction levels commonly higher than those of GC-HRMS [13, 15, 17, 18,19], while for the dl-PCB fraction, CALUX values are often lower than those obtained by
GC-HRMS [17, 18, 19]. However, in this study, only the non-ortho PCBs were measured by
GC-HRMS, while the CALUX assay can detect any PCB (including non-ortho and monoortho) that can bind to and activate the AhR. Although mono-ortho PCBs have low TEF/
REP values, this could maybe partly explain the higher BEQ values found for the PCB
fraction with the CALUX-bioassay compared to GC-HRMS.

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The concentrations of all PCDD/F and non-ortho PCB congeners (pg WHO-TEQ/sample)
and the recoveries of all the C13 standards (%), measured with GC-HRMS, are presented inTable 5. Spiking of the serum samples with the C13 standards was done before extraction of
the sample via the CALUX procedure. The % recovery in Table 5 therefore reflects therecovery of the total CALUX sample pre-treatment method (extraction and clean up) after
measurement with GC-HRMS.

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All congeners could be quantified by GC-HRMS in the pooled serum sample. The recoveryof the C13 spiked PCDD/Fs was between 60–90 % for all congers and between 67–82 % for
the C13 spiked non-ortho PCBs. Also, an adequate separation between both fractions was
obtained during the clean up of the sample extract. In the PCB fraction, not more than 13%of the dioxin congener TCDF (WHO 2005-TEF of 0.1 ) was found which corresponds to0.01 pg CALUX-BEQ / g fat and 9.4% of the dioxin congener TCDD (WHO 2005-TEF of1), which corresponds to 0.81 pg BEQ/g fat. In the PCDD/F fraction not more than 14% of
the PCB congener 169 (WHO 2005-TEF of 0.03) or 0.83 pg CALUX-BEQ/ g fat was
detected, while for the most potent PCB congener, PCB 126 with a WHO 2005-TEF valueof 0.1, 8.6% or 1.0 pg BEQ/g fat was found in the PCDD/F fraction (Table 5).
3.6 Quantification of PCDD/Fs and dioxin-like PCBs in human serum samples fromadolescents$watermark-text

Five mL serum samples obtained from 173 14–15 year-old adolescents of the general
population, recruited via the school system in Flanders, were analyzed using the optimized
CALUX procedure described above. The dl-PCB and PCDD/F fraction were measured
separately for all samples with the H1L7.5c1 cell line, using dilution factors for thesefractions of respectively 2.4 and 5. For these dilution factors, quantification of the sample
extracts could be carried out on 100% and 98.8% of the PCDD/F and dl-PCB fractions
respectively. The PCDD/F fraction of 45 out of 173 samples were also analyzed using theH1L6.1c3 cell line (also at df 5) to compare the two cell lines. The % RSD was lower than
20% for 42 out of 45 samples (see supplementary info: Table 7).
The geometric mean PCDD/F and dioxin-like PCB concentrations in the total study
population (n=173) were respectively 108 (95% confidence interval (CI): 101–114) pg
CALUX-BEQ/g fat and 32.1 (95% CI: 30.1–34.2) pg CALUX-BEQ/g fat. The levels of bothbiomarkers were significantly higher for samples from boys (124.9 pg CALUX-BEQ/g fat
for PCDD/Fs and 36.7 pg CALUX-BEQ/g fat for dl-PCBs) than for those of girls (92.6 pg
CALUX-BEQ/g fat for PCDD/Fs and 27.9 pg CALUX-BEQ/g fat for dl-PCBs) (ANOVA:
p<0.001). The PCDD/F CALUX-BEQ value increased with a lower education level(p=0.02) and was significantly higher in subjects consuming self-caught fish (p=0.02). For
dl-PCBs only a non-significant increasing trend was seen for some food factors such as
eating self-caught fish (p=0.12) and local eggs (p=0.17) and being breastfed as newborn(p=0.07).

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4. Conclusions and discussion
An optimized method was validated for the quantification of PCDD/Fs and dioxin-like
PCBs in small amounts of human serum samples. With the newly developed sensitive
H1L7.5c1 cell line, it was possible to detect both dioxin like-PCBs and PCDD/Fs in serum
samples of adolescents and adults.

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First, full dose-response dilution curves of a pooled sample were established and hence, an
optimum dilution range of the sample matrix for analysis with the CALUX cell line wasobtained. The choice of an appropriate dilution factor is very important, since matrix effects
can occur when dosing concentrated sample extracts and this could result in inaccurate BEQ
determinations. Normally, it should be expected that all dilutions yield the same CALUXBEQ result. However, when the slope and the maximum of the sample curve are not thesame as those of the standard TCDD calibration curve, a wide spread in calculated BEQ
values can be found for different dilutions (Table 4). Therefore, it should be investigatedwhich dilution range yields comparable CALUX-BEQ values. Often this optimum dilution
range is around the EC50 value, when using the mouse hepatoma cell lines. For the serum
samples dilution factors of 2.4 for dl-PCBs and 5 for PCDD/Fs were selected for routine
analysis, since these dilutions yielded CALUX-BEQ results with a same order of magnitude
that were around the EC50 value. All individual serum samples were then analyzed withthese dilution factors.

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These results show the need to establish a full dose curve before starting routine analysis todetermine the optimum dilution range. A single point analysis can not provide the needed
information, since one does not know if this point is in the “toxic range” of the full dose
curve (i.e. past the top of the curve) or not. When the optimum dilution range for the(biological) sample matrix is known, a single point analysis can be done for time-saving andeconomic reasons and/or if not enough sample is available for measuring multiple dilutions,which may be the case for human serum samples which should be used sparingly.

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The validation studies showed that the relative standard deviation for the repeatability andwithin-lab reproducibility for the QC standard were lower than the in-house quality control
criteria limits of 15 and 20 %. For the pooled serum samples a % RSD for long-term withinlab reproducibility of 41% for the dl-PCB fraction and 25% for the PCDD/F fraction, both
expressed as pg BEQ/g fat, was found. The mean recovery of the procedural blanks was forboth fractions within the accepted limits of 80–120%. The LOQ was 14.5 pg BEQ/g fat forthe dl-PCBs and 30.3 pg BEQ/g fat for the PCDD/Fs, taking into account a mean fat amountin the 5 mL serum sample of 0.0165 g for adolescents and the used total dilution factors. The
mean EC50 values for the H1l7.5c1 and H1L6.1c3 cell lines were respectively 438.6 and1804.5 fg/well. The recovery, measured by analysis of a C13-spiked pooled serum sample
with GC-HRMS, was between 60–90% for all PCDD/F congeners and between 67–82% for
the non-ortho PCBs. The separation between both fractions was also adequate: a maximumof 13% of PCDD/F congeners was found in the PCB fraction and maximum 14% of PCB
congeners was found in the PCDD/F fraction. CALUX values from the pooled serum
samples were higher than concentrations calculated with GC-HRMS, with a CALUX/GCHRMS ratio of respectively 2.0 and 1.4 for the PCDD/Fs and the dl-PCBs. As expected,
given the similarity in the two recombinant cell lines, a good correlation was found between
the samples of adolescents analyzed using both the H1L7.5c1 and the H1L6.1c3 cell lines.
The CALUX-BEQ values, reported for the adolescents in this study, were 108 (95% CI:
101–114) pg CALUX-BEQ/g fat for the PCDD/Fs and 32.1 (30.1–34.2) CALUX-BEQ/g fat
for the dioxin-like PCBs. These results will be used as a reference value for Flanders andwill allow the researchers to determine the pollution pressure in specific hot spots and to

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Croes et al.

Page 9follow up the concentration levels in adolescents over a period of time. Comparison of the
CALUX-BEQ values with the results of other instrumental and bio-analytical studies will be
more difficult, since the biological response of the CALUX assay is strongly dependent on
the cell type and the measurement conditions.

$watermark-text

The PCDD/F values in this study were relatively high compared to most values from humanbiomonitoring studies that have been reported in literature (Table 6), but it is not always
known which sample analysis protocol was followed in the other studies. Often different
techniques are used (CALUX rat cells, CALUX mouse cells, GC-HRMS and analysis withor without separation of PCDD/Fs and dioxin-like PCBs), which makes clear interpretationand comparison of the results difficult. Also differences between clean up methods [19], theused dilution factor employed, different methods for quantification using CALUX standard
curves [20] and the choice of TEF/REP values for quantification with GC-HRMS [21] all
have an important influences on the quantification of the final result. Therefore, it is difficult
to compare different studies with different protocols unless a correlation between the two
methods has been established. This can be done by analyzing the same pooled sample(s),with a concentration(s) in the expected range of the unknown samples, multiple times withboth methods.

Supplementary Material
Refer to Web version on PubMed Central for supplementary material.

$watermark-text

Acknowledgments
This study was commissioned, financed and steered by the Ministry of the Flemish Community (Department of
Economics, Science and Innovation; Flemish Agency for Care and Health; and Department of Environment, Natureand Energy).
The authors are grateful to M. Denison and the other members of the University of California-Davis (USA) for
generously providing our laboratory with the H1L6.1c3 and the H1L7.5c1 cell lines, which were developed withfunding from a Superfund Research Program grant (ES04699) from the National Institute of Environmental Health
Sciences. The authors also acknowledge K. Servaes, B. Van den Bosch and M. Wevers for the GC-HRMS analysis(VITO, Unit Environmental Analysis and Technology, Mol, Belgium).

References$watermark-text

1. Long M, Andersen BS, Lindh CH, Hagmar L, Giwercman A, Manicardi G-C, Bizzaro D, Spanò M,
Toft G, Pedersen HS, Zvyezday V, Bonde JP, Bonefeld-Jorgensen EC. Environmental Health: A
Global Access Science Source. 2006; 5:14–27. [PubMed: 16725033]
2. Van Larebeke N, Covaci A, Schepens P, Hens L. Journal of Epidemiology and Community Health.
2002; 56:828–830. [PubMed: 12388572]
3. VMM reports PCDD/Fs and PCB 126 in atmospheric deposition samples (Dutch). http://
www.vmm.be/lucht/meetresultaten/dioxines-en-pcbs-resultaten (09/03/2011).
4. Website of the Flemish human biomonitoring study (FLEHS). www.milieu-en-gezondheid.be/
English/index.html (09/05/2011)
5. Denison MS, He G, Baston DS, Tsutsumi T. Organohalogen Compounds. 2008; 70:772–775.
6. He G, Tsutsumi T, Zhao B, Baston DS, Zhao J, Heath-Pagliuso S, Denison MS. Toxicological
Sciences. 2011 submitted.
7. Conner KT, Harris MA, Edwards MR, Budinsky RA, Clark GC, Chu AC, Finley BL, Rowlands JC.
Journal of Exposure Science and Environmental Epidemiology. 2008; 18:369–380. [PubMed:
17912254]
8. Leng J-H, Kayama F, Wang P-Y, Nakamura M, Nakata T, Wanga Y. Chemosphere. 2009; 75:634–
639. [PubMed: 19211123]

Talanta. Author manuscript; available in PMC 2012 December 17.

Croes et al.

Page 10$watermark-text$watermark-text$watermark-text

9. Warner M, Eskenazi B, Patterson DG Jr, Clark G, Turner WE, Bonsignore L, Mocarelli P, Gerthoux
PM. Journal of Exposure Analysis and Environmental Epidemiology. 2005; 15:310–318. [PubMed:
15383834]
10. Chou I-C, Lee W-J, Wang L-C, Chang-Chien G-P, Lee W-S, Lee H. Journal of Hazardous
Materials. 2008; 154:1166–1172. [PubMed: 18191892]
11. Vanderperren H, Van Wouwe N, Behets S, Windal I, Van Overmeire I, Fontaine A. Talanta. 2004;
63:1277–1280. [PubMed: 18969559]
12. Schroijen C, Van Wouwe N, Sanctorum H, Goeyens L, Baeyens W. Organohalogen Compounds.
2006; 68:2511–2514.
13. Van Wouwe N, Windal I, Vanderperren H, Eppe G, Xhrouet C, Massart A-C, Debacker N, Sasse
A, Baeyens W, De Pauw E, Sartor F, Van Oyen H, Goeyens L. Talanta. 2004; 63:1157–1167.
[PubMed: 18969545]
14. Windal I, Van Wouwe N, Eppe G, Xhrouet C, Debacker V, Baeyens W, De Pauw E, Goeyens L.
Environ. Sci. Technol. 2005; 39:1741–1748. [PubMed: 15819233]
15. Croes K, Van Langenhove K, Elskens M, Desmedt M, Roekens E, Kotz A, Denison MS, Baeyens
W. Chemosphere. 2011; 82(5):718–724. [PubMed: 21094512]
16. Van den Berg M, Birbaum LS, Denison MS, De Vito M, Farland W, Feeley M, Fiedler H,
Hakansson H, Hanberg A, Haws L, Rose M M, Safe S, Schrenk D, Tohyama C, Tritscher A,
Tuomisto J, Tysklind M, Walker N N, Peterson RE. Toxicological Sciences. 2006; 93(2):223–241.
[PubMed: 16829543]
17. Tsutsumi T, Amakura T, Nakamura M, Brown DJ, Clark GC, Sasaki K, Toyoda M, Maitani T.
Analyst. 2003; 128:486–492. [PubMed: 12790202]
18. Van Overmeire I, Carbonnelle S, Schoeters G, Van Cleuvenbergen R, Windal I, Van Wouwe N,
Goeyens L. Organohalogen Compounds. 2003; 60:243–246.
19. Van Wouwe N, Windal I, Goeyens L. Organohalogen Compounds. 2007; 69:817–820.
20. Elskens M, Baston DS, Stumpf C, Haedrich J, Keupers I, Croes K, Denison MS, Baeyens W,
Goeyens L. Talanta. 2011
21. Van Overmeire I, Clark GC, Brown DJ, Chu MD, Cooke M, Denison MS, Baeyens W, Srebrnik S,
Goeyens L. Environmental Science & Policy. 2001; 4:345–357.
22. Koppen G, Covaci A, Van Cleuvenbergen R, Schepens P, Winneke G, Nelen V, Schoeters G.
Toxicology Letters. 2001; 123:59–67. [PubMed: 11514106]
23. Kayama F, Horiguchi H, Oguma E, Fujino J, Yabushita H, Brown D, Clark G. Organohalogen
compounds. 2002; 55:275–278.
24. Todaka T, Hirakawa H, Kajiwara J, Hori T, Tobiishi K, Yasutake D, Onozuka D, Sasaki S,
Miyashita C, Yoshioka E, Yuasa M, Kishi R, Iida T, Furue M. Chemosphere. 2010; 78:185–192.
[PubMed: 19850319]
25. Wittsiepe J, Fürst P, Schrey P, Lemm F, Kraft M, Eberwein G, Winneke G, Wilhelm G.
Chemosphere. 2007; 67:S286–S294. [PubMed: 17217986]
26. Burns JS, Williams PL, Sergeyev O, Korrick S, Lee MM, Revich B, Altshul L, Patterson DG Jr,
Turner WE, Needham LL, Saharov I, Hauser R. Environ Health Perspect. 2009; 117:1593–1599.
[PubMed: 20019911]
27. Ayotte P, Dewailly E, Lambert GH, Perkins SL, Poon R, Feeley M, Larochelle C, Pereg D.
Environ Health Perspect. 2005; 113:1318–1324. [PubMed: 16203240]

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Page 11$watermark-text$watermark-text

Figure 1.

2,3,7,8-TCDD standard calibration curves for the mouse hepatoma H1L6.1c3 and theH1l7.5c1 cell lines. The working range is indicated by the arrows.

$watermark-text
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Croes et al.

Page 12$watermark-text
Figure 2.

$watermark-text

Full dose-response curves for the PCDD/F fraction from the same pooled serum samples,
measured with the H1L7.5c1 cell line (experiment in duplicate). The percentage induction(RLUs relative to the maximum of the TCDD standard curve) is shown at the positions
indicated by the arrows.

$watermark-text
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Page 13

Table 1$watermark-text

CALUX-BEQ values in pg/g fat for the PCDD/F fraction of two full dose response curves from pooled serum
samples. The numbers in bold indicate the optimum dilution range that can be used for quantification. The
results were not corrected for blank responses.
Dilution factor

pg CALUX-BEQ/ g fat PCDD/F fraction
Curve 1

1.2

Curve 2
10.6

$watermark-text

1.5

11.7

15.9

2

16.9

36.7

4

66.2

80.3

5

82.5

71.9

8

102

97.7

20

174

165

31

226

199

40

271

219

$watermark-text
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Table 2$watermark-text

Overview of the repeatability, within-lab reproducibility and apparent recovery for the QC standard, measured
with the H1l7.5c1 cell line. SD is standard deviation; RSD is the relative standard deviation, also often called
coefficient of variation (CV). SDr and RSDr describe the repeatability; SDRW and RSDRW show the withinlab reproducibility.
H1L7.5c1$watermark-text

spike QC standard (pg/well)

0.250

number of plates/days

30

mean QC value (pg/well)

0.245

SDr (pg/well)

0.016

RSDr (%)

6.7

SDRW (pg/well)

0.028

RSDRW (%)

11.4

Recovery (%)

98$watermark-text
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Croes et al.

Page 15

Table 3Long-term within-lab reproducibility of a pooled serum sample, analyzed with the H1L7.5c1 cell line

$watermark-text

PCB fraction

PCDD/F fraction

pg BEQ/ g fat

pg BEQ /g serum

pg BEQ/ g fat

pg BEQ /g serum

Mean

20.0

0.09

83.3

0.40

SD

8.3

0.04

20.8

0.14

% RSD

41

42

25

34

n

35

35

51

51

$watermark-text$watermark-text
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Table 4$watermark-text

Ratio between CALUX and GC-HRMS for the PCDD/F and PCB fractions of a pooled serum sample, where nequals the number of analysis. TEF-values were from 2005 [16]. Only non-ortho PCBs were analyzed with
GC-HRMS.
PCDD/Fs

PCBs

Sum PCDD/Fs anddl-PCBs

GC-HRMS (pg WHO-TEQ/g fat)

41.7

14.7

56.4

CALUX (pg BEQ/g fat)

83.3 (n=51)

20.0 (n=35)

103.3

Ratio CALUX/GC-HRMS

2.0

1.4

1.8

$watermark-text$watermark-text
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Table 5$watermark-text

GC-HRMS congener concentrations (pg WHO-TEQ/sample) and C13 standard recoveries (%) from CALUX
sample extraction and clean up for PCDD/Fs and dl-PCBs. TEF-values were from 2005 [16]. nd is notdetected.

$watermark-text

Dioxin fraction

Dioxin fraction

PCB fraction

PCB fraction

pg WHO-TEQ% recovery

pg WHO-TEQ% recovery

2,3,7,8-T4CDF

0.0080

60.6

nd

13

1,2,3,7,8-P5CDF

0.0051

80.5

nd

0.7

2,3,4,7,8-P5CDF

1.7

89.7

nd

2.5

1,2,3,4,7,8-H6CDF

0.16

79.8

nd

0.6

1,2,3,6,7,8-H6CDF

0.17

75.1

nd

0.4

2,3,4,6,7,8-H6CDF

0.045

74.6

nd

0.1

1,2,3,7,8,9-H6CDF

0.0080

63.3

nd

0.2

1,2,3,4,6,7,8-H7CDF

0.021

69.2

nd

0.1

1,2,3,4,7,8,9-H7CDF

0.00070

77.6

nd

0.1

GC-HRMS
PCDD/Fs$watermark-text

O8CDF

0.00012

60.8

nd

0.1

2,3,7,8-T4CDD

0.99

67.5

nd

9.4

1,2,3,7,8-P5CDD

2.0

88.8

nd

1.3

1,2,3,4,7,8-H6CDD

0.15

79.9

nd

0.3

1,2,3,6,7,8-H6CDD

1.5

75

nd

0.8

1,2,3,7,8,9-H6CDD

0.14

-

nd

1,2,3,4,6,7,8-H7CDD

0.11

71.8

nd

0.2

O8CDD

0.039

59.5

nd

0.1

PCB 81nd

4.4

0.0001168.9

PCB 77nd

4.9

0.0002167.2

PCB 126

nd

8.6

1.7

82.1

PCB 169

nd

13.7

0.75

72.5

dl-PCBs

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$watermark-text$watermark-text
2000

BelgiumTalanta. Author manuscript; available in PMC 2012 December 17.

172

341

Students (14–15
years old), general
population
Adults, men and
women

1999
1999

Flanders, Peer
Flanders, Antwerp

Japan

Japan
Germany
RussiaCanadaTodaka et al.,2010 [24]Wittsiepe et al.,2007 [25]Burns et al., 2009[26]

Ayotte et al.,2005 [27]na2003–2005

2000–2003

2002–2005

2002

1999

2002

Flanders, Antwerp

2002

SwedenUkraine1999

Adults, men

2002

PolandFlanders, Peer

Adults, men

2002–2004

Greenland

482

40Adults, men andwomen, 25–75
years old

169

Children 8–9
years old

Pregnant women,
19–42 years old

119

1407

Female farmers,
55,5 years old(average)
Mothers25

Adults, women,
50–65 years old

25

Adults, women,
50–65 years old
22

22Adults, women,
50–65 years old

Adults, women,
50–65 years old

86

78

99

75

Adults, men

Adults, men

2000

341

173

Students (14–15
years old), general
population

Adults, men and
women

N

Population

Belgium

Kayama et al.,2002 [23]Koppen et al.,2001 [22]Long et al., 2006
[1]

Van Wouwe etal., 2004 [13]2008–09

Flanders

This Study

2008–09

Flanders

This Study

PeriodCountry

ReferenceMedian (min-max)

MedianMean

Mean (SD)

Mean (SD)GM (95%CI)

GM (95%CI)

Mean (SD)

Mean (SD)medianmedianmedianmedianGMGM

GM (95%CI)

GM (95%CI)

Unit

pg CALUX TEQ/g fat

pg WHO-TEQ/g fat

pg WHO-TEQ/g fat

pg WHO-TEQ/g fat

pg CALUX TEQ/g fat

pg WHO-TEQ/g fat

pg WHO-TEQ/g fat

pg CALUX TEQ/g fat

pg CALUX TEQ/g fat

pg CALUX TEQ/g fat

pg CALUX TEQ/g fat

pg CALUX TEQ/g fat

pg CALUX TEQ/g fat

pg WHO-TEQ/g fat

pg CALUX TEQ/g fat

pg CALUX-BEQ/g fat

pg CALUX-BEQ/g fat

CALUX in pg TEQ/g fat

Calculation

Value

102 (37–287)

21.1

16.79 PCDD/F
11.57 dl-PCB

11(4.2) PCDD/F
5.5 (2.5) dl-PCB

32.3 (12.1)

78.9 (72.7–85.6)

70.9 (65.3–76.9)

35.0 (16.5)

37.2 (13.1)

337

428

312

197

25.7

41.8

32.1 (30.1–34.2)

108 (101–114)

Literature overview for the PCDD/F concentrations from human biomonitoring studies in different countries

BDS-CALUX, sum PCDD/F and dlPCB

GC-HRMS

GC-HRMS

GC-HRMS

XDS-CALUX, PCDD/F

GC-HRMS, sum PCDD/F and dl-PCB

GC-HRMS, sum PCDD/F and dl-PCB

BDS-CALUX, sum PCDD/F and dlPCB

BDS-CALUX, sum PCDD/F and dlPCB

UCD-CALUX, Hepa1.12cR

UCD-CALUX, Hepa1.12cR

UCD-CALUX, Hepa1.12cR

UCD-CALUX, Hepa1.12cR

GC-HRMS, PCDD/F

XDS-CALUX, H1L6.1c2 PCDD/F

UDC-CALUX H1L7.5c1, dl-PCB

UDC-CALUX, H1L7.5c1 PCDD/F

Method$watermark-text

Table 6
Croes et al.
Page 18

ItalyWarner et al.,2005 [9]1999

Women, 20–49
years old

Population
22

N$watermark-text
PeriodMean (min-max)

Calculation
pg CALUX TEQ/g fat

Unit$watermark-text

Country
30.8 (1.6–67.3)

Value
XDS-CALUX, PCDD/F

Method$watermark-text

Reference

Croes et al.
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