CALUX DETERMI ATIO OF PCDD/F’S A D DIOXI -LIKE PCB’S I SMALLAMOU TS OF HUMA MILK FROM THE RURAL AREAS OF FLA DERS(BELGIUM) USI G THE H1L7.5C1 MOUSE HEPATOMA CELL LI E
Croes K.1,5,Vandermarken T.1, Colles A.2, Bruckers L.3, Koppen G.2, Govaerts E.2 , Nelen V.4, Van de Mieroop
E.4, Schoeters G.2, Van Larebeke N.5, Denison M.S.6 , Kotz A.7, Van Langenhove K.1, Elskens M.1 and BaeyensW.1
1Vrije Universiteit Brussel, Department of Analytical and Environmental Chemistry (ANCH), Pleinlaan 2, 1050
Brussels, Belgium; 2Vito, Environmental Health and Risk, Boeretang 200, 2400 Mol, Belgium; 3Interuniversity
Institute for Biostatistics and Statistical Bioinformatics, Hasselt University, Belgium; 4Provincial Institute for
Hygiene, Antwerp, Belgium; 5Ghent University Hospital, Study Centre for Carcinogenesis and Primary
Prevention of Cancer, Ghent, Belgium; 6Department of Environmental Toxicology, University of California,Davis, CA 95616, USA; 7 State Institute for Chemical and Veterinary Analysis of Food, Bissierstrasse 5,Freiburg, Germany
Introduction
In the first Flemish Environment and Health survey run by the Flemish Centre of Expertise on Environment andHealth (FLEHS I 2002-2006) increased concentrations of PCBs, dioxin-like substances and chlorinatedpesticides (a metabolite of DDT and hexachlorobenzene) were observed in cord blood of newborns and in bloodof 14-15 year-old adolescents and 50-65 year-old adults living in low populated rural communities of East andWest Flanders and Flemish Brabant compared to other Flemish regions. Due to the health concern associatedwith chlorinated compounds follow-up of pollutant levels in this area is of importance. Therefore, human breast
milk from mothers living in these regions was collected (2009-2010) for analysis of different POPs.
For the quantification of PCDD/Fs and/or dioxin-like PCBs in (human) milk samples both GC-HRMS and theCALUX bioassay are used in routine analysis. However, most CALUX methods use high amounts of milk(varying between 10 and 60 mL)1, 2. Since for the Flemish human breast milk survey in the rural areas not only
PCDD/F and dioxin-like PCBs were analyzed, but also other POPs like DDE, PBDEs, marker PCBs, HCB,HCH, perfluorinated compounds, etc., the amount of milk available for the CALUX bioassay was limited.
Therefore, a new method was developed for the analysis of PCDD/Fs and dioxin-like PCBs in only 5 mL milk.
Materials and methods
In this study, 5 mL human breast milk samples obtained from 84 18-35 year-old breastfeeding mothers residingin the rural areas of Flanders (24 low-populated rural communities in East and West Flanders), wereanalyzed for PCDD/Fs and dl-PCBs with a new sensitive CALUX mouse hepatoma cell line. Additionally, fromeach of the 84 individual samples, 10 mL was taken to compose a pooled sample, which was analyzed with both
the CALUX assay (VUB, ANCH, Brussels, Belgium) and by GC-HRMS (WHO reference laboratory, State
Institute for Chemical Analysis and Veterinary of Food, Freiburg, Germany). Details about inclusion criteria
and characteristics of the study population are described by Colles et al., 20113.
Since the matrix of human breast milk is very similar to that of human serum4, the same CALUX protocol wasused for the analysis of the individual human milk samples, but with a higher amount of acid silica due to thehigher fat content of milk compared to blood. Briefly, the milk samples were extracted with 15 mL acetone and3 times 5 mL hexane and the extract was purified on a celite column. The fat extract was weighted andredissolved in hexane. The sample was then further purified on an acid silica column coupled in series with acarbon column.
The cell line used in the bioassay was the sensitive H1L7.5c1 recombinant mouse hepatoma cell line, stablytransfected with pGudLuc 7.5 and containing 5 dioxin responsive domains (5 DRDs)5,6. Cell treatment andmeasurement were based on the protocols described by Windal et al. (2005)7 and the XDS method 4435 from2008. 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 sigmoid dose-response

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curve obtained with 2,3,7,8-TCDD standards. Three quality control (QC) solutions (i.e. a standard solution of
TCDD corresponding to a RLU induction of around 50%) and 3 DMSO blanks were added in duplicate to every
96-well plate as an internal control.
Methods used for analysis of the pooled milk sample by the WHO reference laboratory (State Institute for
Chemical Analysis of Food) in Freiburg, Germany, are described elsewhere8, 9,10.
Results and discussion:
When analyzing a new sample matrix with the CALUX bioassay, it is important to determine the optimum
dilution factor by analysis of different sample dilutions from the same sample or the same pool of samples. In
this study, dilutions curves were obtained for human breast milk samples from 1) a volunteer and 2) from a poolof human breast milk from 5 volunteers.
For the PCB fraction, dilution factors of 1.2; 1.54; 2.4 and 4 were analyzed. Dilution factor 1.2 gave the highest
results for the pooled milk samples, but for the volunteer a maximum response was found for dilution factor
1.54. Dilution factors 2.4 and 4 yielded low % RLU inductions, around the LOQ. Dilution factor 1.54 also gave
the best repeatability (curves 1 and 2 from pool in Figure 1) and therefore, it was decided to use this dilution
factor for further experiments.

Figure 1: Full dose-response curve for the dl-PCB fraction from pooled milk samples, measured with theH1L7.5c1 cell line. The circle indicates the optimum dilution factor.
For the PCDD/F fraction, 4 full dose-response curves with dilution factors 1.2; 1.54; 2; 4; 8; 20 and 40 wereanalyzed. For the pooled human milk samples (curves 1 and 2 from pool on Figure 2), dilution factors 2 and 4
gave % RLU inductions around the EC50 value (inductions between 63.4 and 46.8%). For the dilution curves ofthe volunteer (curves 1 and 2 in dashed line), the optimum dilution factor was 2 (% RLU induction close to theEC50 value and good agreement between the two sample extracts).
Since for the PCDD/F fraction the optimum dilution factor is dependent on the sample load and thus can differ
from one person to another, it was decided to analyze two dilutions for every sample (df 2.5 and 4). For heavily
contaminated samples dilution factor 4 gave values around the EC50 value, while for less contaminated samples
dilution factor 2.5 was better. When the EC50 value was between the % RLU inductions measured for dilution
factor 2.5 and 4, an extrapolation to the EC50 was made.

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Figure 2: Full dose-response curve for the PCDD/F fraction from pooled milk samples, measured with theH1L7.5c1 cell line. The circles indicate the optimum dilution factors
The geometric mean of the individual samples in the total study group (n=84) was 10.4 (95% CI: 9.4-11.4) pg
CALUX-BEQ/g fat for the PCDD/Fs and 1.73 (1.57-1.91) pg CALUX-BEQ/g fat for the dl-PCBs. PCDD/F
concentrations were significantly higher for mothers who lost weight compared to mothers who recently gained
weight (p=0.012) and also for the consumption of local eggs (p=0.023). For both PCDD/Fs and dl-PCB, asignificant increase in BEQ was found with a higher amount of breast milk fat (p=12E-5 for PCDD/Fs and
p=0.007 for dl-PCBs).
The CALUX-BEQ values on the pooled breast milk sample were in good agreement with the GC-HRMS datafor both the PCDD/F and the dl-PCB fractions (Table 1). The PCDD/F BEQ values were 32 % and 60 % higher
with CALUX compared to GC-HRMS, when using respectively the 1998 and 2005 TEF scheme. For the dlPCBs a better agreement was found between the two techniques with the 2005 TEF scheme (a CALUX/GCHRMS ratio of 0.58 compared to 0.37 for the 1998 TEFs).
pg WHO TEQ/g fat
GC-HRMS
GC-HRMS
MeanRatio: meanand(TEF 1998)(TEF 2005)
CALUX-BEQ CALUX/GC-HRMS
pg BEQ/ g fat(n=2)(TEF 1998/2005)
8.41
6.95
11.09
1.32/1.60
PCDD/Fs
5.80
3.77
2.17
0.37/0.58
dl-PCBs
14.21
10.72
13.26
0.93/1.24
Total dl-compounds
Table 1: The CALUX-BEQ and GC-HRMS WHO-TEQ values, expressed per g of fat, for the PCDD/F
and dl-PCB fractions from a pooled human milk sample. TEF values from 1998 and 2005 were used.
The GC-HRMS TEQ values on the pooled human milk sample (Table 1) were lower than the Belgian result
from the 4th WHO-coordinated survey of human milk for persistent organic pollutants (2006). In this WHOstudy respectively 10.31 and 7.02 pg WHO-TEQ/g fat (TEF 1998) was found for the PCDD/F and dl-PCB
fractions11.
The concentration of dl-PCBs in human milk was much lower than these of the PCDD/Fs, which was also foundin former Belgian studies. The PCDD/F levels were comparable to the concentrations found in German studies

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from Wittsiepe et al., 2007 (2000-2003, 13.30 pg WHO-TEQ/ g fat)12 and Raab et al., 2007 (2005: 9.91 pg
WHO-TEQ/g fat)13 and to the European mean concentration found in the 3rd and 4th WHO human breast milksurvey (8.9 pg WHO-TEQ/g fat, 2001 and 2006). When comparing the Belgian PCDD/F levels to the resultsfrom other European countries participating the 4th WHO human milk survey from 2006 (Czech Republic,Slovakia, Sweden, Norway, Finland and Hungary), the Belgian levels were still higher despite the declining
trend3, 14. The dl-PCBs levels were lower in Flanders than in other European studies, where values of 13.00(200-2003)12, 9.92 (2005)13 and 9.4 pg WHO-TEQ/ g fat (mean concentration from 3rd and 4th WHO humanbreast milk campaign, 2001 and 2006) were found.
Acknowledgements:
The authors acknowledge all mothers who kindly accepted to participate to this study, as well as thefieldworkers, midwifes, gynecologists, doctors and directors of the participating maternities, whose cooperation
was essential for the successful results of this study.
This study was commissioned, financed and steered by the Environment, Nature and Energy Department of theFlemish government.
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 H1L7.5c1 cell lines which was developed with funding from aSuperfund Research Program grant (ES04699) from the National Institute of Environmental Health Sciences.
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