1476-069X-6-32 1476-069X Research <p>AhR transcriptional activity in serum of Inuits across Greenlandic districts</p> Long Manhai ml@mil.au.dk Deutch Bente bd@mil.au.dk Bonefeld-Jorgensen C Eva ebj@mil.au.dk

Unit of Cellular and Molecular Toxicology, Department of Environmental and Occupational Medicine, Institute of Public Health, University of Aarhus, Denmark

Centre for Arctic Environmental Medicine, Department of Environmental and Occupational Medicine, Institute of Public Health, University of Aarhus, Denmark

 Environmental Health 1476-069X 2007 6 1 32 http://www.ehjournal.net/content/6/1/32 17956617 10.1186/1476-069X-6-32 13 12 2006 23 10 2007 23 10 2007 2007 Long et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract

Background

Human exposure to lipophilic persistent organic pollutants (POPs) including polychlorinated dibenzo-p-dioxins/furans (PCDDs/PCDFs), polychlorinated biphenyls (PCBs) and organochlorine pesticide is ubiquitous. The individual is exposed to a complex mixture of POPs being life-long beginning during critical developmental windows. Exposure to POPs elicits a number of species- and tissue-specific toxic responses, many of which involve the aryl hydrocarbon receptor (AhR). The aim of this study was to compare the actual level of integrated AhR transcriptional activity in the lipophilic serum fraction containing the actual POP mixture among Inuits from different districts in Greenland, and to evaluate whether the AhR transactivity is correlated to the bio-accumulated POPs and/or lifestyle factors.

Methods

The study included 357 serum samples from the Greenlandic districts: Nuuk and Sisimiut (South West Coast), Qaanaaq (North Coast) and Tasiilaq (East Coast). The bio-accumulated serum POPs were extracted by ethanol: hexane and clean-up on Florisil columns. Effects of the serum extract on the AhR transactivity was determined using the Hepa 1.12cR mouse hepatoma cell line carrying an AhR-luciferase reporter gene, and the data was evaluated for possible association to the serum levels of 14 PCB congeners, 10 organochlorine pesticide residues and/or lifestyle factors.

Results

In total 85% of the Inuit samples elicited agonistic AhR transactivity in a district dependent pattern. The median level of the AhR-TCDD equivalent (AhR-TEQ) of the separate genders was similar in the different districts. For the combined data the order of the median AhR-TEQ was Tasiilaq > Nuuk ≥ Sisimiut > Qaanaaq possibly being related to the different composition of POPs. In overall, the AhR transactivity was inversely correlated to the levels of sum POPs, age and/or intake of marine food.

Conclusion

i) We observed that the proportion of dioxin like (DL) compounds in the POP mixture was the dominating factor affecting the level of serum AhR transcriptional activity even at very high level of non DL-PCBs; ii) The inverse association between the integrated serum AhR transactivity and sum of POPs might be explained by the higher level of compounds antagonizing the AhR function probably due to selective POP bioaccumulation in the food chain.

1. Background

Being resistant to both biotic and abiotic degradation, most persistent organic pollutants (POPs) bioaccumulate and magnify in animals and humans 12. The polychlorinated dibenzo-p-dioxins/furans (PCDDs/PCDFs), polychlorinated biphenyls (PCBs) and organochlorine pesticides are dominating among the POPs. Some POPs e.g. PCBs and persistent organochlorine pesticides, that still are released into the environment due to ongoing use in developing countries and improper storage or disposal in developed countries, can be transported to the Arctic regions by atmospheric and oceanic currents and have been found to bioaccumulate and biomagnify in the Arctic marine food web 34. The traditional diet of the indigenous people in the Arctic largely depends on fish, seabirds and marine mammals being associated with extraordinary high POP exposure 5678910, which may possess a health risk 11. Since 1994, organochlorines have been measured in fat and plasma samples taken from Inuits of Greenland indicating that the contaminant level in Greenland is very high 5891012. It has been documented that exposure to POPs such as dioxins (e.g. 2,3,7,8-tetrachlorodibenzo-p-dioxin, TCDD) and dioxin-like (DL) compounds including non-ortho and mono-ortho PCBs may cause a series of negative effects both in animals and humans including carcinogenicity 13, immunotoxicity and adverse effects on reproductive and neurobehaviour 14. Most of the toxic and biological effects of dioxins and DL-compounds is mediated mainly through activation of the aryl hydrocarbon receptor (AhR), an intracellular ligand-dependent transcriptional factor expressed in most tissues of mammals 15. Upon receptor-ligand binding and translocation into the nucleus, the complex with the AhR nuclear translocator (ARNT) binds to the DNA dioxin-responsive elements (DREs), causing induction of gene transcription, for instance, encoding for metabolic enzymes 16.

Since dioxins and DL-compounds exist as complex mixtures of various congeners in environmental and biologic samples, the concept of TEQ (TCDD toxic equivalent) has been introduced to simplify risk assessment and regulatory control 117. The classical TEQs (WHO-TEQ) are calculated by multiplying the concentration of individual PCDDs/PCDFs/PCBs by their respective Toxic Equivalency Factors (TEFs), which correspond to the relative potency of the congener to generate AhR-mediated effects compared to TCDD, the most potent AhR ligand 117. Studies have emphasized that assessment of the toxicological potential of a chemical mixture is much more complex than can be deduced by a calculated TEQ value 71819. There are several drawbacks using the TEF concept for risk assessment of mixtures of POPs such as expensive and time consuming gas chromatography mass spectrometry (GC-MS) determinations, small concentrations of hardly detectable individual congeners, presence of compounds not routinely measured or unknown substances with AhR affinity, the lack of TEF values for several POPs, and possible antagonistic or synergistic interactions between POPs 2192021. Therefore, there is a need of simple, inexpensive and specific assays that can be used to give an integrated risk assessment of dioxins and DL-compounds. Rapid and sensitive AhR-based screening bioassays are valuable to more fully characterize the ligand-binding specificity of the AhR, to identify AhR ligands and AhR interfering compounds present in the extracts of environmental and biologic samples. A number of bioassays for the determination of TEQ values have been developed 22. The experts have agreed that the use of in vitro bioassays provides an useful tool as a pre-screening method for TEQs in environmental samples 123. The in vitro AhR mediated

    c
hemical
    a
ctivated
    lu
ciferase gene e
    x
pression (CALUX) bioassay is a reporter-gene based cell bioassay using genetically modified cells responding to AhR ligands. The CALUX bioassay has proven to be a quick and sensitive assay to detect the AhR mediated potential of pure chemicals 2202124, extracts of environmental and biological matrices eliciting the biological integrated net TEQ value (CALUX-TEQ) of complex mixtures as found in sediment, pore water 25, bovine and human milk 1926, human serum 27 and follicular fluid 28. Compared to the calculated chemical-derived TEQ (WHO-TEQ), the CALUX based AhR-TEQ might be more biological relevant for the risk assessment of dioxins and DL-compounds 29.

As a part of the human health program of the "Arctic Monitoring and Assessment Program (AMAP)"30, the aim of the present study was i) to compare the AhR transcriptional activity of the actual mixture of lipophilic POP of serum of subjects from different districts in Greenland and ii) to evaluate whether the AhR transcriptional activity is associated to the profile and level of POPs and/or lifestyle factors.

2. Methods

2.1. Subjects, sampling and POPs determination

The subjects and sampling methods have been described in detail elsewhere 10. Briefly, the participants were of Inuit descent from Nuuk, Sisimiut (South West Greenland), Qaanaaq (North West Greenland) and Tasiilaq (East Greenland). The men data of Sisimiut was also a part of the EU project INUENDO. Venous blood samples and questionnaires about demographic and lifestyle factors were collected. The serum was prepared as described 10 and then stored at -80°C until analyzed.

As described 10 plasma was analyzed for POPs including cis-chlordane, trans-chlordane, oxychlordane, p,p'-DDE, p,p'-DDT, hexachlorobenzene (HCB), beta-Hexachlorocyclohexane (β-HCH), mirex, toxaphene 26, toxaphene 50 and 14 PCB congeners (CB28, CB52, CB99, CB101, CB105, CB118, CB128, CB138, CB153, CB156, CB170, CB180, CB183, CB187; in bold are DL-PCB) by gas chromatography (GC). The samples enrolled per district were enough for comparison of the POP levels across districts with a power of 80% in power calculation. Plasma lipids were measured using conventional enzymatic methods and total lipids were calculated as described 31. The lipid adjusted POP data (μg/kg lipid) was accomplished by dividing the wet-weight concentration in plasma (μg/l) by the individual samples lipid concentration and multiplying by 1000 to convert from gram to kilogram 1031. Because of high POP intercorrelation 10 (Bonefeld-Jorgensen and Long, submitted), the group-variables were used for evaluation of the data, namely, the sum of 14 PCB congeners (ΣPCB_14), the sum of pesticides (Σ pesticide) and finally the sum of all the determined POPs (Σ POP).

The fatty acid profiles were determined in plasma phospholipids at the Biology Department, University of Guelph, Canada 32. The n-3 polyunsaturated fatty acids were reported on the sum of C18:3, n-3, C20:4, n-3, C20:5, n-3, C22:5, n-3 and C22:6, n-3, and the n-6 fatty acids was the sum of C18:2, n-6, C18:3, n6, C20:2, n-6, C20:3, n-6 and C20:4, n-6 10. The ratio between n-3 and n-6 is known to be a strong indicator of marine food intake and thus a good indicator of the relative consumption of traditional versus imported food 833.

2.2. AhR transcriptional activation assay

The serum was extracted with a mixture of ammonium sulphate/ethanol/hexane (1:1:3) and the lipid extract was concentrated and cleaned up on two Florisil columns to get the actual mixture of POPs 2731. The detailed descriptions of dissolving the extract samples and detecting serum AhR transcriptional activity has been described in detail elsewhere 27. The stably transfected mouse hepatoma cell line Hepa1.12cR cells carrying the AhR-luciferase reporter gene was kindly provided by MS Denison (University of California, Davis, CA, USA). Similar with Ziccardi and co-workers 34, the optimal conditions in our assay for determination of AhR transcriptional activity of samples using Hepa1.12cR cell line were in 100 μl medium containing 10% FCS with 6 × 104 cells, final solvent concentration of 0.1–0.4% DMSO and 4 h of exposure. Further validation of our Hepa1.12cR bioassay was obtained by participating in two international programs of inter-laboratory comparisons of dioxin-like compounds (The second round of interlaboratory comparison of dioxin-like compounds in food using bioassays, 2004, Örebro University, Sweden, and Interlaboratory comparison of dioxins and dioxin-like compounds in food using CALUX bioassay, 2006, Scientific Institute of Public Health, Belgium). The limit of detection was 64 fg TEQ/well. The Hepa1.12cR cells were exposed for 4 hours to the serum extract alone (termed AhRag) or to mimic physiological environment in the presence of half maximum effective concentration of TCDD (termed AhRcomp) 27. The luciferase activity was determined using a luminometer (BMG LUMISTAR) and cell protein was accomplished using a fluorometer (Wallac 1420 Multilabel Counter, Perkin Elmer life science, FIN) as previously described 35. The luciferase activity of samples was presented as relative light units per microgram protein (RLU/μg protein) 35. The average coefficient of variation (CV) between replicates (intra CV) and between day-to day independent assays (inter CV) of the solvent control was 9% and 17%, respectively and the intra CV of the test samples was 10%.

No cell toxicity on Hepa1.12cR cells was observed after exposure to the serum extract samples determined as described 35.

2.3. Calculation and statistical analysis

In the separate assays the activity differences between the triple serum extract determinations and their respective solvent controls [% agonist (samples with agonistic effect), % inhibition (samples with decrease of TCDD induced activity), and % increase (samples with further increase of TCDD induced activity)] were evaluated using the Student t-test with statistical significant level of p ≤ 0.05 (Microsoft Excel).

The AhR-TEQs values of serum extract were obtained by interpolation of the AhRag values onto the parallel TCDD dose-response sigmoid Hill curve and only the AhRag values being significantly higher than the solvent control and in the linear range of the TCDD dose-response curve were used to calculate AhR-TEQ 27.

Natural logarithmic transformation of AhR-TEQ/AhRcomp and POP data improved the normality (checked by Q-Q plots) and homogeneity of variance, and the statistical analysis was performed on the ln-transformed data. The comparisons of different variables (POPs, AhR-TEQ and AhRcomp) among the districts were performed with One-way ANOVA test. When ANOVA showed statistical significant differences complementary multiple comparison ad hoc tests was performed. Test for equal variances was performed using Levene's test. The least-significant difference (LSD) test was used if the variables showed equal variance; otherwise Dunett T3 test was used. The comparison of variables between the genders was performed by independent Student t-test.

Bivariate correlations were evaluated by Pearson correlation analyses.

The overall associations between the AhR-mediated activities and POPs across the study groups and/or across genders were assessed by comparing the regression lines for each study group/gender by using multiple regression analysis.

Up to date few studies on AhR transcriptional activity in human serum have been reported 2736373839, and thus the knowledge is limited about which dietary or other lifestyle determinants might affect serum AhR transcriptional activity. Our hypothesis is that potential determinants of POP bioaccumulation might also be potential determinants for serum AhR transcriptional activity. Guided by the literature 40, age and seafood intake are known determinants affecting the POP serum level, and intake of seabird might as well influence the POP level. The n-3 fatty acids being determinants of seafood intake 8910, and lifestyle factors such as smoking and BMI might also influence the serum POP level 8. Using the multiple linear regression model, assessing the relation between the POPs and AhR activities, the impact of potential confounders were evaluated by entering variables together with ΣPCB_14, Σ pesticide and Σ POP as follows: in the first step, age and then seafood intake (represented by n-3/n-6 ratio) were included in the model, then additionally smoking years and BMI were included in the model. Finally, the bird intake was additionally included in the model. The potential confounder was defined if the regression coefficient (β) changes more than 10% and when a significant association (p ≤ 0.05) was obtained after adjustment. Then the calculations were adjusted for the confounders.

The inter-district variations in serum levels of POPs, AhR-TEQ and AhRcomp were also assessed by linear regression models and age was in these models considered as a potential confounder of these variables.

The statistical analysis was performed on SPSS 13.0 (SPSS Inc. Chicago, IL, USA). The statistical significant level was set to p = 0.05.

3. Results

3.1. The demographical and lifestyle factors of study groups

As reported 10, the age of the participants was from 18–77 with the median age of 35 years old. The Nuuk men were significantly older than the other participants, whereas the age range of the remaining male and female participants of all four districts were similar (Table 1). No significant difference was observed for the BMI of the participants from all districts. For the smoking years, Nuuk men and women had the highest median, whereas the other three districts were similar for both sexes (Table 1). The Nuuk and Sisimiut men had the highest and the lowest median n-3/n-6 ratio, respectively, whereas for women significantly higher n-3/n-6 ratio was observed for Qaanaaq and Tasiilaq. The median seabird intake of the Nuuk men was the highest whilst for the rest of subject's similar median seabird intake was observed (Table 1). Nuuk and Sisimiut participants had higher median consumption of dairy food than that of Qaanaaq and in overall male participants had lower dairy food intake than women (Table 1).

<p>Table 1</p>

Basic characteristics of study population

Men

Nuuk

Sisimiut

Qaanaaq

Tasiilaq

All

Age (years)

n

50

52

43

41

186

median

54*

30

34

34

37

mean

54

31

34

34

39

min-max

35–77

18–46

19–45

19–45

18–77

BMI (kg/m2)

n

46

52

43

41

182

median

28

26

27

26

26

mean

28

27

27

26

27

min-max

22–35

19–36

20–41

21–33

19–41

Smoking (years)

n

45

52

41

40

178

median

34*

12

18

15

18

mean

31

12

17

15

19

min-max

0–59

0–26

0–31

0–31

0–59

n-3/n-6

n

50

37

43

41

171

median

0.61*

0.22▲

0.40

0.40

0.40

mean

0.74

0.29

0.48

0.45

0.50

min-max

0.20–1.93

0.10–0.73

0.09–1.45

0.12–1.03

0.09–1.93

Seabird intake (per month)

n

47

49

41

na

137

median

2.0*

1.0

1.0

na

2.0

mean

5.5

2.6

2.6

na

3.6

min-max

0–20

0–28

0–20

na

0–28

Dairy food consumption (per month)

n

48

51

43

na

142

median

41

51

32*♠

na

41

mean

39

49

30

na

40

min-max

2–84

8–84

3–58

na

2–84

Women

Age (years)

n

45

42

36

48

171

median

38

31

34

29

34

mean

36

33

33

30

33

min-max

19–45

18–44

18–44

19–45

18–45

BMI (kg/m2)

n

43

42

36

48

169

median

26

26

24

25

25

mean

26

27

26

26

26

min-max

18–32

19–47

18–34

17–40

17–47

Smoking (years)

n

42

42

34

48

166

median

20

12

16

12

14

mean

16

13

16

13

14

min-max

0–33

0–27

0–29

0–31

0–33

n-3/n-6

n

45

38

36

48

167

median

0.25

0.29

0.40#

0.41#

0.33

mean

0.29

0.32

0.48

0.46

0.38

min-max

0.12–0.72

0.14–0.82

0.15–1.53

0.18–1.0

0.12–1.53

Seabird intake (per month)

n

44

41

35

na

120

median

1.0

1.0

1.0

na

1.0

mean

1.4

1.3

3.4

na

1.9

min-max

0–8.0

0–2.0

1.0–28

na

0–28

Dairy food consumption (per month)

n

45

42

35

na

122

median

56

57

37

na

49

mean

53

49

36

na

47

min-max

2–84

0–84

0–84

na

0–84

Men + Women

Age (years)

n

95

94

79

89

357

median

44

31

34

32

35

mean

46

31

33

32

36

min-max

19–77

18–46

18–45

19–45

18–77

BMI (kg/m2)

n

89

94

79

89

351

median

26

26

26

25

26

mean

27

27

27

26

26

min-max

18–35

19–47

18–41

17–40

17–47

Smoking (years)

n

87

94

75

88

344

median

23

12

17

13

16

mean

23

12

16

14

16

min-max

0–59

0–27

0–31

0–31

0–59

n-3/n-6

n

95

75

79

89

338

median

0.36

0.25

0.40

0.40

0.36

mean

0.51

0.30

0.48

0.45

0.44

min-max

0.12–1.93

0.10–0.82

0.09–1.53

0.12–1.03

0.09–1.93

Seabird intake (per month)

n

91

90

76

na

257

median

2.0

1.0

1.0

na

1.0

mean

3.5

2.0

3.0

na

2.8

min-max

0–20

0–28

0–28

na

0–28

Dairy food consumption (per month)

n

93

93

78

na

264

median

48

51

36

na

43

mean

46

49

33

na

43

min-max

2–84

0–84

0–84

na

0–84

na: not available.

*: p < 0.05 versus other districts; ▲: p < 0.05 versus Nuuk and Sisimiut men; #: p < 0.05 versus Nuuk and Sisimiut women; ♠: p < 0.05 versus Nuuk and Sisimiut men and all women.

3.2. The plasma level of POPs

For the men, similar median levels of Σ PCB_14, Σ pesticide and Σ POP were observed among Nuuk, Qaanaaq and Tasiilaq and all were significantly higher than that of Sisimiut with the order of Nuuk ≥ Qaanaaq ≥ Tasiilaq > Sisimiut. Tasiilaq and Qaanaaq women had significantly higher POPs median levels than that of Nuuk and Sisimiut women (Table 2) as reported 10. In general, male participants had higher median POP levels than women in all districts. For the combined sex data, Tasiilaq and Qaanaaq had similar POPs median levels which were significantly higher than that of Nuuk and Sisimiut participants (Tasiilaq ≥ Qaanaaq > Nuuk > Sisimiut). After adjustment for age, the pattern of regional difference did not change (data not shown).

<p>Table 2</p>

The plasma POP levels in the subjects of the different district

Men

Nuuk

Sisimiut

Qaanaaq

Tasiilaq

All

Σ PCB_14 (μg/kg lipid)

n

38

51

41

37

167

median

2682

540.9*

1854

1890

1526

mean

3094

752.1

2699

2079

2057

min-max

490–11113

132–2366

617–7352

318–4527

132–11113

Σ pesticide (μg/kg lipid)

n

38

51

41

41

171

median

3401

737.8*

2630

2242

2126

mean

4034

1012

3824

2372

2684

min-max

405–19118

141–3542

617–14366

194–7224

141–19118

Σ POP (μg/kg lipid)

n

38

51

41

37

167

median

6322

1353*

4411

4067

3551

mean

7127

1765

6524

4249

4692

min-max

896–30231

285–5908

1234–19807

499–11260

285–30231

ΣDL-PCB/Σ PCB_14 (%)

n

38

51

41

37

167

median

8.42♠

7.07

7.53

7.51

7.70

mean

8.36

7.41

7.44

7.73

7.71

min-max

5.0–11.0

4.0–11.0

4.0–11.0

6.0–11.0

4.0–11.0

Women

Σ PCB_14 (μg/kg lipid)

n

44

42

34

47

167

median

295.7

346

1188▲

1660▲

635.5

mean

492.4

459.7

1406

1809

1041

min-max

97.5–2428

67.2–1384

99–5980

112–6701

67.2–6701

Σ pesticide (μg/kg lipid)

n

44

42

34

48

168

median

481.6

496.3

1671▲

2044▲

860

mean

678

700

2018

2397

1446

min-max

83.6–2881

75.6–3014

77.9–7529

153–6995

85.5–7529

Σ POP (μg/kg lipid)

n

44

42

34

47

167

median

748

823

2847▲

3797▲

1469

mean

1171

1159

3424

4169

2480

min-max

181–5309

143–4337

177–12432

256–12829

143–12829

ΣDL-PCB/Σ PCB_14 (%)

n

44

42

34

47

167

median

9.01

9.26

8.45

8.96

9.00

mean

9.02

9.40

8.78

9.02

9.10

min-max

4.0–13.0

6.0–15.0

6.0–13.0

6.0–12.0

4.0–15.0

Men + Women

Σ PCB_14 (μg/kg lipid)

n

82

93

75

84

334

median

912.4

495.3

1379#

1806#

1068

mean

1698

620.0

2113

1928

1549

min-max

97.5–11113

67.2–2366

99–7352

112–6701

67.2–11113

Σ pesticide (μg/kg lipid)

n

82

93

75

89

339

median

1336

685.3

2156#

2208#

1327

mean

2234

871

3006

2386

2071

min-max

83.6–19118

75.6–3542

77.9–14366

153–7224

85.5–19118

Σ POP (μg/kg lipid)

n

82

93

75

84

334

median

2322

1244

3437#

4020#

2420

mean

3931

1491

5119

4206

3597

min-max

181–30231

143–5908

177–19807

256–12829

143–30231

ΣDL-PCB/Σ PCB_14 (%)

n

82

93

75

84

334

median

8.79

7.92

7.88

8.40

8.30

mean

8.71

8.31

8.05

8.45

8.39

min-max

4.0–13.0

4.0–15.0

4.0–13.0

6.0–12.0

4.0–15.0

Σ PCB_14: sum of 14 PCB congeners; Σ pesticide: sum of 10 pesticides; Σ POP: Σ PCB_14 + Σ pesticide; ΣDL-PCBs: sum of dioxin-like PCBs (PCB105, PCB118, PCB156).

*: p < 0.05, versus men from other districts; ▲: p < 0.05, versus Nuuk and Sisimiut women;

#: p < 0.05, versus Nuuk and Sisimiut subjects; ♠: p < 0.05, versus men from other districts.

The dioxin-like PCBs (DL-PCBs) determined in this study were CB105, CB118 and CB156. The Nuuk men had higher median percentage of ΣDL-PCB of the Σ PCB_14 than men from other districts (Nuuk > Tasiiaq ≥ Qaanaaq ≥ Sisimiut). No significant difference of ΣDL-PCB percentage between women from different districts was observed but a lower trend was observed for Qaanaaq women (Table 2). Women had significantly higher median percentage of DL-PCBs than men. In overall, the median percentage of ΣDL-PCB for the combined data was in the order of Nuuk ≥ Tasiilaq ≥ Sisimiut > Qaanaaq (Table 2).

3.3. AhR-TEQ and AhRcomp activity

91% of all male and 79% of all female serum samples showed significant agonistic AhR transcriptional activity. The levels of AhR-TEQ differed significantly among the districts for the separate gender (Table 3, Fig. 1A &1B). The order of median level of AhR-TEQ was Tasiilaq ≥ Sisimiut ≥ Nuuk > Qaanaaq for men (Fig. 1A), and for women Nuuk ≥ Tasiilaq ≥ Sisimiut > Qaanaaq (Fig. 1B). Thus for both genders Qaanaaq had a lower AhR-TEQ level than participants from other districts (Table 3).

<p>Table 3</p>

The serum levels of AhR transcriptional activity of subjects of the different district

Men

Nuuk

Sisimiut

Qaanaaq

Tasiilaq

All

AhR-TEQ1 (pg/g lipid)

n

21

42

38

38

139

median

166

189

110*

240

153

mean

206

247

118

296

219

min-max

95 – 1019

26 – 1202

58 – 271

98 – 1452

26 – 1452

% agonist

79

88

100

97

91

AhRcomp2 (RLU/μg protein)

n

38

51

43

38

170

median

0.78*

1.21

1.35

1.04

1.15

mean

0.82

1.24

1.38

1.12

1.15

min-max

0.38 – 1.18

0.59 – 2.46

0.77 – 2.17

0.31 – 3.24

0.31 – 3.24

% increase

0

44

54

29

33

% inhibition

32

4

2

32

16

Women

AhR-TEQ1 (pg/g lipid)

n

12

32

32

44

120

median

235

188

122*

228

167

mean

326

221

131

332

248

min-max

115 – 1055

93 – 1032

63 – 259

100 – 1987

63 – 1987

% agonist

64

64

100

91

79

AhRcomp2 (RLU/μg protein)

n

45

42

36

44

167

median

1.41

1.33

1.31

1.28

1.32

mean

1.50

1.38

1.33

1.92

1.54

min-max

0.9 – 4.54

0.94 – 3.88

0.74 – 1.87

0.25 – 15

0.25 – 15

% increase

49

64

58

46

54

% inhibition

0

0

11

21

8

Men + Women

AhR-TEQ1 (pg/g lipid)

n

33

74

70

82

259

median

188

189

115

239

161

mean

250

236

124

315

232

min-max

94 – 1055

26 – 1202

58 – 271

98 – 1987

26 – 1987

% agonist

71

76

100

94

85

AhRcomp2 (RLU/μg protein)

n

83

93

79

82

337

median

1.08

1.26

1.33

1.18

1.23

mean

1.19

1.30

1.36

1.55

1.35

min-max

0.38 – 4.54

0.59 – 3.88

0.74 – 2.17

0.25 – 15

0.25 – 15

% increase

27

53

56

38

43

% inhibition

15

2

6

26

12

1AhR-TEQ (TCDD equivalents): The AhR-TEQ of samples eliciting significantly agonistic activity was calculated by interpolation to the TCDD dose-response curve using the Sigmaplot program, given as pg/g serum lipid. The % agonistic indicates the % of samples eliciting a significant increase in AhR activity compared to the solvent control. 2AhRcomp: AhR competitive activity of serum extract + 60 pM TCDD (EC50) given as RLU/μg protein. EC50 solvent control = 1 RLU/μg protein; % increase and % inhibition indicates the % of samples responding with a further increase or decrease of the EC50(TCDD) induced activity, respectively. *: p < 0.05 versus subjects from other districts.

 <p>Figure 1</p>

The serum AhR transcriptional activity

The serum AhR transcriptional activity. A) The AhR-TEQ in men from different districts; B) The AhR-TEQ in women from different districts; C) The AhRcomp activity in men from different districts and D) The AhRcomp activity in women from different districts. The reference line of the control is given as dash line (C, D). The values in the figure are percentage of sample with agonistic (A, B) and increase or inhibition of TCDD induced AhR transcriptional activity (C, D).

AhRcomp activity was assessed by co-exposure with serum extract and 60 pM TCDD (EC50(TCDD)). The median AhRcomp activity of men was in the order of Qaanaaq ≥ Sisimiut ≥ Tasiilaq > Nuuk, reflecting the higher percentage of samples elicited inhibition of the TCDD induced AhR activity in the serum samples of Nuuk and Tasiilaq (Table 3, Fig. 1C). Higher percentage of serum samples of men from Qaanaaq and Sisimiut elicited a further increase of the TCDD induced AhR activity (Table 3, Fig. 1C). The serum samples of women from the four districts had similar AhRcomp level; mainly eliciting a further increase of the TCDD induced AhR activity (Table 3, Fig. 1D).

No significant difference of the AhR-TEQ level was found between the genders in the separate district (Table 3). In the Nuuk study group, men had a lower level of AhRcomp than women, whereas similar AhRcomp levels were found for the two genders for Sisimiut, Qaanaaq and Tasiilaq participants (Table 3).

Adjustment for age did not change the regional and gender difference of AhR-TEQ and AhRcomp (data not shown).

3.4. The association between AhR-TEQ, AhRcomp and POPs

3.4.1. The correlations between the single POPs

Pearson correlation analyses showed that most PCB congeners were significantly and mutually intercorrelated (r > 0.82; p < 0.001), whereas CB 28, CB52, CB128 and CB101 had a relatively lower correlation coefficients to other PCB congeners (r = 0.29–0.82; p < 0.05). Also the determined pesticides were intercorrelated (r > 0.75 – 0.99). The grouping variables (ΣPCB_14 and Σ pesticide) intercorrelated with r = 0.97 and the sum of determined POPs as Σ POP was therefore acceptable.

3.4.2. The correlations between AhR-TEQ, AhRcomp and POPs

3.4.2.1. Multiple regressions of AhR-TEQ and AhRcomp on POPs across the study groups and genders

Multiple regression analysis of the combined districts and genders data was performed to analyze for homogeneity/heterogeneity of the associations between POPs and AhR-TEQ/AhRcomp (see Additional file 1). The analyses indicated that the associations of POPs and AhR-TEQ were allowed to be evaluated in each district for the combined gender, whereas the AhRcomp activity and POPs association must be evaluated in each single district for men and for women in combined districts.

<p>Additional file 1</p>

Multiple regressions of the combined study groups. The data provided represents the statistical analysis of the homogeneity/heterogeneity of the associations between POPs and AhR-TEQ/AhRcomp among the districts and genders.

Click here for file

However, in order to obtain the overall trend of the studied groups and a better statistical power, the data based on the combined genders of the single districts for POPs and AhRcomp and the combined genders across the districts were also evaluated for the correlation of POPs and AhR-TEQ/AhRcomp.

3.4.2.2. Correlations between AhR-TEQ, AhRcomp and POPs

An inverse correlation between AhR-TEQ and POPs was observed for the combined men data and the total combined data (Table 4). Few significant correlations between AhR-TEQ and POPs were found for the single districts for the two sexes. Significantly inverse correlations between AhR-TEQ and the POP variables were observed for both the Qaanaaq women and the combined Qaanaaq participants before and after adjustment for age and/or n-3/n-6 (Table 4).

<p>Table 4</p>

Multivate linear regression analysis of AhR-TEQ and POPs

Nuuk

Sisimiut

Qaanaaq

Tasiilaq

All

Mena

n

β

p

n

β

p

n

β

p

n

β

p

n

β

p

Σ PCB_14

raw1

21

-.01

.97

41

-.24

.13

37

-.14

.41

33

-.06

.72

135

-.21

.01

+age

21

.05

.86

41

-.09

.64

37

.05

.80

33

.03

.87

135

-.16

.11

+n-3/n-62

21

-.01

.98

29

-.25

.25

37

-.30

.23

33

-.11

.54

123

-.22

.07

+age+n-3/n-6

21

.07

.87

29

-.06

.81

37

-.14

.60

33

.01

.98

123

-.16

.21

Σ pesticide

raw

21

.08

.72

41

-.27

.087

37

-.20

.23

37

-.13

.46

139

-.25

<.01

+age

21

.15

.57

41

-.14

.44

37

-.01

.98

37

-.05

.78

139

-.21

.04

+n-3/n-62

21

.16

.61

29

-.34

.13

37

-.49

.06

37

-.20

.23

127

-.37

<.01

+age+n-3/n-6

21

.22

.53

29

-.20

.39

37

-.30

.32

37

-.09

.60

127

-.31

.01

Σ POP

raw1

21

.04

.86

41

-.26

.10

37

-.19

.24

33

-.11

.56

135

-.16

.01

+age

21

.11

.69

41

-.12

.50

37

-.01

.96

33

-.02

.93

135

-.20

.06

+n-3/n-62

21

.09

.78

29

-.30

.17

37

-.48

.07

33

-.15

.40

123

-.29

.02

+age+n-3/n-6

21

.16

.66

29

-.15

.54

37

-.29

.31

33

-.04

.82

123

-.23

.06

Womenb

Σ PCB_14

raw1

12

.06

.85

32

.23

.20

32

-.46

.01

43

-.09

.55

119

-.04

.70

+age

12

-.01

.99

32

.25

.24

32

-.40

.05

43

-.21

.35

119

-.002

.99

+n-3/n-62

12

.58

.19

28

.17

.43

32

-.32

.18

43

-.13

.50

115

.06

.62

+age+n-3/n-6

12

.53

.31

28

.20

.38

32

-.25

.36

43

-.23

.35

115

.08

.53

Σ pesticide

raw1

12

.11

.74

32

.25

.16

32

-.53

<.01

44

-.11

.46

120

-.06

.52

+age

12

.06

.89

32

.26

.19

32

-.50

.01

44

-.28

.20

120

-.03

.74

+n-3/n-62

12

.69

.12

28

.19

.39

32

-.51

.04

44

-.17

.35

116

.01

.97

+age+n-3/n-6

12

.65

.20

28

.20

.37

32

-.47

.09

44

-.31

.19

116

.02

.89

Σ POP

raw1

12

.09

.79

32

.24

.18

32

-.51

<.01

43

-.11

.49

119

-.05

.57

+age

12

.03

.95

32

.25

.22

32

-.48

.02

43

-.24

.28

119

-.02

.83

+n-3/n-62

12

.66

.14

28

.17

.42

32

-.47

.07

43

-.15

.43

115

.04

.77

+age+n-3/n-6

12

.62

.23

28

.20

.39

32

-.42

.14

43

-.27

.27

115

.05

.67

Men + Womenc

Σ PCB_14

raw1

33

-.19

.29

74

-.05

.65

70

-.32

.01

77

-.10

.40

254

-.15

.02

+age

33

-.09

.79

74

.03

.83

70

-.22

.01

77

-.07

.65

254

-.10

.18

+n-3/n-62

33

.12

.72

58

-.15

.30

70

-.34

.02

77

-.15

.24

238

-.16

.06

+age+n-3/n-6

33

.24

.60

58

-.09

.53

70

-.26

.09

77

-.11

.49

238

-.12

.17

Σ pesticide

raw1

33

-.13

.47

74

-.07

.58

70

-.38

<.01

82

-.12

.28

254

-.18

<.01

+age

33

.06

.84

74

.01

.94

70

-.30

.03

82

-.12

.38

254

-.13

.07

+n-3/n-62

33

.29

.37

58

-.16

.29

70

-.47

<.01

82

-.21

.09

238

-.24

<.01

+age+n-3/n-6

33

.41

.29

58

-.12

.44

70

-.40

.02

82

-.17

.22

238

-.21

.02

Σ POP

raw1

33

-.16

.38

74

-.06

.59

70

-.37

<.01

77

-.12

.30

254

-.17

.01

+age

33

-.001

.997

74

.01

.92

70

-.29

.04

77

-.10

.49

254

-.12

.09

+n-3/n-62

33

.23

.50

58

.16

.28

70

-.45

<.01

77

-.18

.17

238

-.20

.02

+age+n-3/n-6

33

.36

.395

58

-.11

.46

70

-.31

.02

77

-.14

.36

238

-.16

.07

The analyses were performed on ln transformed data with AhR-TEQ as dependent and POPs as independent variables before and upon adjusted for the potential confounders.

1: non-adjusted data.

2: ratio of n-3 to n-6 fatty acids in serum.

a: Combining districts is not allowed according to the multiple regression analyses.

b: Combining districts is not allowed according to the multiple regression analyses.

c: According to the multiple regression analyses, combining sex for each district is allowed while combining sexes for all districts is not allowed.

For Σ PCB_14, Σ pesticide and Σ POP, AhR-TEQ, see the legend of Table 2 and Table 3

As shown in Table 5, the total combined data and the combined Tasiilaq data showed an inverse relationship between AhRcomp and POPs before and after adjustment for age and n-3/n-6. The significantly inverse correlations of AhRcomp and POPs observed for all men data disappeared upon adjustment for age and/or n-3/n-6 ratio (Table 5). Also for the combined Nuuk data, negative correlations of AhRcomp and POPs were observed, which disappeared upon adjustment for age plus n-3/n-6 ratio (Table 5). For the two sexes in the single districts, few significant correlations between AhRcomp and POPs were observed but Tasiilaq women showing an inverse correlation upon adjustment for age and/or n-3/n-6. In overall, negative association between AhRcomp and POP data was found but Sisimiut men where AhRcomp activity was positively related to the determined ΣPCB_14 upon adjustment for age and n-3/n-6.

<p>Table 5</p>

Multivate linear regression analysis of AhRcomp and POPs

Nuuk

Sisimiut

Qaanaaq

Tasiilaq

All

Mena

n

β

p

n

β

p

n

β

p

n

β

p

n

β

p

Σ PCB_14

raw1

38

-.11

.52

51

-.13

.36

41

-.07

.68

34

-.18

.31

164

-.24

<.01

+age

38

-.16

.47

51

-.03

.87

41

-.001

1.00

34

-.29

.15

164

-.05

.56

+n-3/n-62

38

-.24

.37

37

.29

.12

41

.08

.68

34

-.20

.27

150

-.01

.95

+age+n-3/n-6

38

-.30

.34

37

.42

.04

41

.11

.61

34

-.29

.15

150

.10

.35

Σ pesticide

raw1

38

-.09

.59

51

-.20

.17

41

-.12

.46

38

-.27

.11

168

-.24

<.01

+age

38

-.10

.59

51

-.12

.45

41

-.07

.75

38

-.36

.05

168

-.06

.53

+n-3/n-62

38

-.16

.52

37

.22

.27

41

.01

.97

38

-.29

.10

154

-.04

.73

+age+n-3/n-6

38

-.16

.52

37

.29

.17

41

.04

.87

38

-.36

.06

154

.07

.49

Σ POP

raw1

38

-.10

.55

51

-.18

.22

41

-.10

.53

34

-.19

.28

164

-.24

<.01

+age

38

-.13

.53

51

-.09

.57

41

-.04

.84

34

-.30

.13

164

-.06

.53

+n-3/n-62

38

-.20

.43

37

.25

.20

41

.04

.86

34

-.21

.25

150

-.02

.87

+age+n-3/n-6

38

-.22

.43

37

.34

.10

41

.07

.76

34

-.30

.13

150

.09

.46

Womenb

Σ PCB_14

raw1

44

.08

.60

42

17

.28

34

-.31

.07

43

-.18

.24

163

-.09

.24

+age

44

.16

.37

42

.09

.59

34

-.15

.46

43

-.51

.02

163

-.11

.17

+n-3/n-62

44

.18

.25

38

.09

.62

34

-.49

.06

43

-.27

.14

159

-.11

.25

+age+n-3/n-6

44

.25

.14

38

.07

.71

34

-.33

.25

43

-.56

.02

159

-.13

.21

Σ pesticide

raw1

44

.07

.67

42

.09

.57

34

-.25

.15

44

-.21

.18

164

-.11

.18

+age

44

.11

.49

42

.-.001

.995

34

-.07

.72

44

-.55

.01

164

-.13

.13

+n-3/n-62

44

.18

.26

38

-.03

.85

34

-.37

.17

44

-.30

.10

160

-.14

.18

+age+n-3/n-6

44

.22

.19

38

-.06

.76

34

-.19

.52

44

-.59

.01

160

-.15

.15

Σ POP

raw1

44

.08

.62

42

.12

.45

34

-.27

.12

43

-.195

.21

163

-.10

.21

+age

44

.14

.42

42

.03

.85

34

-.10

.64

43

-.54

.01

163

-.12

.15

+n-3/n-62

44

.19

.24

38

.01

.97

34

-.42

.12

43

-.29

.12

159

-.13

.21

+age+n-3/n-6

44

.24

.15

38

-.02

.94

34

-.24

.42

43

-.59

.01

159

-.14

.17

Men + Womenc

Σ PCB_14

raw1

82

-.55

<.01

93

-.05

.64

75

-.14

.22

77

-.22

.06

327

-.24

<.01

+age

82

-.35

.02

93

-.07

.58

75

-.04

.76

77

-.39

.01

327

-.19

<.01

+n-3/n-62

82

-.39

.01

75

-.06

.66

75

-.07

.62

77

-.27

.03

309

-.21

<.01

+age+n-3/n-6

82

-.21

.25

75

-.08

.55

75

-.001

1.00

77

-.41

.01

309

-.18

.02

Σ pesticide

raw1

82

-.53

<.01

93

-.10

.34

75

-.15

.20

82

-.23

.04

332

-.24

<.01

+age

82

-.30

.03

93

-.13

.27

75

-.04

.80

82

-.38

.01

332

-.18

<.01

+n-3/n-62

82

-.34

.02

75

-.12

.35

75

-.08

.61

82

-.28

.02

314

-.21

.01

+age+n-3/n-6

82

-.17

.28

75

-.14

.29

75

.01

.93

82

-.40

<.01

314

-.18

.02

Σ POP

raw1

82

-.54

<.01

93

-.09

.42

75

-.15

.21

77

-.22

.05

327

-.24

<.01

+age

82

-.32

.03

93

-.11

.35

75

-.04

.80

77

-.40

.02

327

-.19

<.01

+n-3/n-62

82

-.37

.01

75

-.10

.43

75

-.07

.62

77

-.27

.03

309

-.21

<.01

+age+n-3/n-6

82

-.19

.28

75

-.13

.35

75

.011

.95

77

-.41

.01

309

-.18

.02

The analyses were performed on ln transformed data with AhRcomp as dependent and POPs as independent variables before and upon adjusted for the potential confounders.

1: non-adjusted data.

2: ratio of n-3 to n-6 fatty acids in serum.

a: Combining districts is not allowed according to the multiple regression analyses.

b: Combining districts is allowed according to the multiple regression analyses.

c: Combining districts and sex is not allowed according to the multiple regression analyses.

For Σ PCB_14, Σ pesticide and Σ POP and AhRcomp, see the legend of Table 2 and Table 3.

Further adjustment for BMI, smoking year and bird intake did not change the pattern of associations of POPs and AhR-TEQ, AhRcomp (data not shown).

Scattered and few significant correlations of AhR-TEQ/AhRcomp and the single PCB and pesticides and/or Σ DL-PCBs were observed (data not shown). Since the congeners of PCBs were highly intercorrelated and the AhR-TEQ represents the integrated activity of the POP mixture, it is hard to assess which of the compounds having the highest impact on the AhR-TEQ/AhRcomp level.

3.5. Correlations between lifestyle factors, POPs and AhR transcriptional activity

3.5.1. POPs

As found earlier 10, for the combined data the levels of ΣPCB_14, Σpesticide and ΣPOPs were positively associated with age (r > 0.449, p < 0.001) and seafood intake represented by n-3/n-6 ratio (r > 0.614, p < 0.001), and age was positively correlated to seafood intake (r = 0.472, p < 0.001).

3.5.2. AhR-TEQ

As shown in Table 6, scattered and few correlations between AhR-TEQ and lifestyle factors were found in the single districts and the separate genders, most of which involved the age, and/or the n-3/n-6 ratio. A significantly negative correlation between age and AhR-TEQ was observed for Sisimiut men and the combined men data. For Sisimiut women, a positive correlation between BMI and AhR-TEQ and for Qaanaaq women a negative correlation of the n-3/n-6 ratio and AhR-TEQ were observed. Significantly negative correlation between age and AhR-TEQ was also found for the combined Qaanaaq data and the total combined data. For the combined Nuuk, Sisimiut and Qaanaaq data a significantly, positive correlation between AhR-TEQ and consumption of dairy food was found (Table 6), suggesting that dairy products may be one source of DL-compounds and/or AhR activating compounds for the Inuits living in West Greenland.

<p>Table 6</p>

The Pearson correlation coefficients of serum AhR-TEQ/AhRcomp and life style factors

Men

Women

Men + Women

AhR-TEQ

AhRcomp

AhR-TEQ

AhRcomp

AhR-TEQ

AhRcomp

Nuuk

n

r

p

n

r

p

n

r

p

n

r

p

n

r

p

n

r

p

age

20

-.08

.74

37

-.02

.91

11

.12

.72

44

-.13

.41

32

-.19

.28

82

-.53

<.01

n-3/n-6

20

-.01

.97

37

-.02

.91

11

-.32

.32

44

-.30

.05

32

-.26

.15

82

-.50

<.01

BMI

19

.40

.08

35

. 24

.15

11

.07

.84

42

-.19

.22

31

.13

.48

78

-.17

.14

smoking

19

.04

.86

32

.12

.52

8

.56

.15

34

.22

.21

27

.13

.53

66

-.22

.08

seabird

19

.001

1.0

34

.01

.94

8

-.47

.20

37

-.09

.56

28

-.19

.32

72

-.43

<.01

diary food

20

-.26

.25

36

-.12

.48

11

.34

.29

44

.014

.93

32

.03

.85

81

.19

.09

Sisimiut

age

41

-.32

.04

50

-.21

.14

31

.08

.65

41

.21

.18

73

-.18

.12

92

.01

.91

n-3/n-6

29

-.06

.75

36

-.31

.06

27

.21

.28

37

.29

.07

57

-.10

.48

74

.08

.55

BMI

41

-.05

.76

50

-.16

.28

31

.52

<.01

41

-.18

.27

73

.19

.10

92

-.16

.14

smoking

35

-.36

.03

43

-.12

.44

29

.11

.56

33

.01

.94

64

-.15

.22

76

-.03

.78

seabird

38

-.01

.97

46

-.12

.43

28

-.17

.38

38

-.05

.74

67

-.05

.72

85

-.12

.28

diary food

40

.15

.37

49

.06

.69

30

-.003

.99

40

.23

.15

71

.09

.44

90

.14

.18

Qaanaaq

age

37

-.28

.08

42

-.11

.48

31

-.33

.07

35

-.34

.05

69

-.30

.01

78

-.21

.66

n-3/n-6

37

-.002

.99

42

-.16

.30

31

-.42

.02

35

-.06

.74

69

-.17

.15

78

-.11

.31

BMI

37

-.02

.49

42

-.08

.60

31

-.11

.54

35

.04

.83

69

-.13

.28

78

-.02

.87

smoking

33

-.12

.49

37

-.12

.49

29

-.23

.22

33

-.35

.05

62

-.20

.12

70

-.21

.08

seabird

35

-.19

.27

39

-.33

.04

30

-.05

.81

34

-.23

.19

66

-.01

.37

74

-.28

.17

diary food

37

-.02

.91

42

.15

.34

29

-.01

.96

35

.14

.42

67

.001

.99

76

.14

.22

Tasiilaq

age

37

-.19

.25

37

.05

.76

43

.04

.81

43

.10

.53

81

-.07

.54

81

.03

.76

n-3/n-6

37

.26

.12

37

.01

.95

43

.12

.90

43

.02

.91

81

.12

.28

81

.007

.95

BMI

37

-.002

.91

37

-.002

.99

43

-.27

.08

43

-.08

.60

81

-.19

.10

81

-.07

.52

smoking

33

.01

.97

33

-.003

.99

41

.06

.72

41

.05

.75

74

.03

.79

74

.02

.90

seabird

na

na

na

na

na

na

na

na

na

na

na

na

na

na

na

na

na

na

diary food

na

na

na

na

na

na

na

na

na

na

na

na

na

na

na

na

na

na

All

age

138

-.19

.03

169

-.35

<.01

119

-.09

.34

166

.03

.66

258

-.16

.01

336

-.20

<.01

n-3/n-6

126

.10

.27

155

-.20

.01

115

-.12

.19

162

-.04

.59

242

-.02

.81

318

-.16

.01

BMI

137

-.02

.86

167

-.01

.77

119

.01

.87

164

-.02

.23

257

-.01

.93

332

-.09

.10

smoking

120

-.17

.07

145

-.23

.01

107

-.01

.96

141

.04

.68

227

-.11

.11

286

-.11

.06

seabird*

94

-.07

.47

121

-.35

<.01

68

-.20

.10

111

-.13

.16

163

-.12

.12

233

-.33

<.01

diary food*

99

.13

.18

129

.02

.79

72

.21

.08

119

.13

.15

172

.17

.03

249

.11

.08

na: not available

*: For the combined data of Nuuk, Sisimiut and Qaanaaq.

3.5.3. AhRcomp activity

The AhRcomp data was also in overall inversely related to age and/or the n3/n6 ratio, but also the seabird intake seemed to be a determinant. A negative correlation for AhRcomp to seabird intake was observed for the Qaanaaq men and all men data (Table 6). Moreover, the AhRcomp of all men was found to be inversely correlated to age, n-3/n-6 ratio and smoking years. Negative correlations between AhRcomp and the n-3/n-6 ratio and age and smoking years were found for the women of Nuuk and Qaanaaq, respectively. For the combined sex data of Nuuk subjects, negative correlations between AhRcomp and age, n-3/n-6 ratio and seabird intake were found (Table 6). By combining all data (districts and sexes), AhRcomp was found to be negatively correlated to age and n3/n6 ratio (Table 6). Besides, seabird intake was found to be negatively correlated to AhRcomp for all men data, the total combined data of Nuuk, Sisimiut and Qaanaaq groups (Table 6).

4. Discussion

In the present study we compared the ex vivo net integrated AhR transcriptional activity of the lipophilic serum POP mixture of Inuits among different Greenlandic districts and evaluated whether the serum AhR transcriptional activity was related to the bioaccumulated POPs and/or lifestyle factors. The serum levels of POPs, AhR-TEQ and AhRcomp activity differed among the two sexes living in different Greenlandic districts (Northwest-Qaanaaq, South West-Nuuk and Sisimiut and East-Tasiilaq). Also the association of the AhR transcriptional activity to the determined POPs differed between the districts and genders. We observed for the combined data an inverse association between AhR-TEQ and POPs where the Qaanaaq women had main influence. The negative association of AhRcomp activity to POPs was mainly contributed by Nuuk and Tasiilaq subjects.

As reported 10, the POPs levels of Tasiilaq and Qaanaaq participants (including men and women) were in general higher than that of Nuuk and Sisimiut subjects, which is consistent with previous reports that inhabitants of East Greenland had higher POP levels than those in the West Greenland 6841 and in accordance with POP levels in the marine biota of North West Greenland and East Greenland being higher than that of the Central West Coast 41. A greater prevalence of western lifestyle habits (consumption of imported food) was reported for Nuuk and Sisimiut subjects 1012, which can explain the lower POP burden in Sisimiut and Nuuk subjects compared to Qaanaaq and Tasiilaq. However, in the present study, Nuuk men elicited higher POP levels than men from other districts, which may be due to their higher age and high intake of seafood and seabird 1042.

The pattern of regional differences of serum AhR-TEQ levels did not follow the district-distribution of POPs levels. The AhR-TEQ level of Nuuk men having the highest POP level was lower than that of Sisimiut men with the lowest POPs level, whereas Qaanaaq women with relatively higher POPs level elicited the lowest AhR-TEQ level. However, the regional differences of AhR-TEQ seemed to mimic the district variation of the ratio of the ΣDL-PCB to the ΣPCB_14. For both sexes, the DL-PCB proportion of Qaanaaq subjects was in general lower than that of Nuuk, Tasiilaq and Sisimiut (men: Nuuk > Tasiilaq ≥ Qaanaaq ≥ Sisimiut; women: Sisimiut ≥ Nuuk ≥ Tasiilaq ≥ Qaanaaq) and consistently lower AhR-TEQ level was observed for Qaanaaq men and women (men: Tasiilaq ≥ Sisimiut ≥ Nuuk > Qaanaaq; women: Nuuk ≥ Tasiilaq ≥ Sisimiut > Qaanaaq). Similarly, male participants in all districts had higher POP burden but lower DL-PCB proportion and accordingly a tendency of lower AhR-TEQ level compared to female was observed in the present study. Moreover, we also found that individuals with higher ΣDL-PCB/Σ PCB_14 ratio tend to have higher AhR-TEQ levels while those with higher Σ POP have significantly lower AhR-TEQ than individuals with lower Σ POP (data not shown). Our study further support the previous report that DL-PCBs were major contributors of the total WHO-TEQ concentration in the plasma of Arctic Inuits 43. Therefore, the total POPs level may not be a suitable proxy of the DL-compounds levels and thus the composition of the POPs in the body must be taken into account for the assessment of the net AhR transcriptional activity of serum samples 27.

Ayotte and coworkers reported that the average plasma WHO-TEQ level of Inuit adults living in Arctic Quebec was 184 pg/g lipid 43, being compatible with the serum AhR-TEQ level of Greenlandic Inuits determined in the present study (161 pg/g lipid). Previously, we reported a lower AhR-TEQ level for Greenlandic Inuit males (197 pg/g lipid) than for European men from Sweden, Warsaw and Kharkiv with the average serum AhR-TEQ level of 317 pg/g lipid 27. The present study further supports that Greenlandic Inuits have lower level of AhR-TEQ than European individuals. The lower level of AhR-TEQ in Inuits compared to Europeans might be due to lower level of DL-compounds and lipophilic AhR activating compounds as well as higher level of AhR inhibiting POPs in the Inuit samples in addition to the report of lower level of PCDD/Fs in the Arctic region versus Europe and America 44. Furthermore, re-evaluation of our previous study 27 by stratifying on the Inuits and European Caucasians showed a significantly positive correlation of POP proxy marker CB-153 and AhR-TEQ in European Caucasians (rs = 0.20, p = 0.001), combined with their higher AhR-TEQ level, suggesting European Caucasians were more close to DL-compounds sources.

Knowing that the measured net integrated AhR induced activity of the lipophilic serum extract are in the linear range (Bonefeld-Jorgensen and Long, submitted), and running a TCDD dose-response measurement in parallel, we suggest that the chemical mixture in serum extract can further increase or inhibit the TCDD induced AhR mediated activity (termed as AhRcomp). In the present study, the AhRcomp differed between districts and between sexes. Compared to Qaanaaq and Sisimiut men, Nuuk and Tasiilaq men had the lowest AhRcomp median level being in accordance with the higher frequency of samples eliciting inhibition of the TCDD induced AhR activity, suggesting the presence of compounds having the potency to compete with TCDD for the AhR activation. The higher incidence of further increase of the TCDD induced effect observed for Sisimiut and Qaanaaq men as well as women from all districts suggest the presence of compounds interacting with TCDD for AhR mediated effects. The differences in the incidence of further increase and inhibition of TCDD induced activity might be caused by the different POP composition of Inuits from the different districts and for genders also differences in diet intake might have an impact on the AhR function 810.

Humans are exposed to a combination of several xenobiotics. It is likely that the interaction between these contaminants, additivity and/or synergy and/or antagonism, will be reflected in the final toxic effect. In vivo and in vitro studies have reported that PCB mixtures and some individual PCB congeners antagonized TCDD effects 4546, and Arochlor 1254 was reported to inhibit TCDD induced 7-ethoxyresorufin O-deethylase (EROD) activity 47. A mixture of PCBs, based on the congeners identified in human milk, significantly decreased TCDD induced rat liver EROD activity 45. For the POPs determined in the serum extract in this study, only 3 DL-PCBs (CB105, CB-118 and CB156) were shown to have agonistic AhR potentials 11723, while other PCBs such as PCB153, PCB 52, PCB128 have the potential to antagonize the AhR pathway 2048 and HCB was also reported to inhibit the TCDD induced activity 49. Chlordane was reported to decrease the basal and TCDD induced CYP 1A1 promoter activity in MCF-7 cells 50. Furthermore, most of the determined pesticides and PCBs in this study were reported as xenoestrogens (p,p'-DDT, p,p'-DDE, toxaphene, chlordane) 51525354 or antiestrogens (CB138, CB153, CB180) 5556. Previous studies showed that o,p'-DDT and other xenoestrogenic pesticides such as endosulfan and dieldrin significantly decreased the basal and TCDD induced AhR mediated gene expression 5057. Moreover, high concentrations of weak agonists can lead to antagonism 2158. Also, there may be other forms of antagonism when there are large concentrations of other compounds relative to high affinity ligands. Non-DL-PCBs can both antagonize and/or synergize the effects of DL-compounds, it depends on the response, the dose, and the concentrations 4859. As humans are exposed to a complex mixture and many PCBs can act as AhR antagonists most likely in concert when present together, this could actually influence the overall effect of DL-compounds by decreasing the net serum AhR transcriptional activity. Since the serum AhR transcriptional activity determined in the present study is the net integrated effect of the AhR agonists and antagonists of the constituents in the mixture, the negative correlation of AhR-TEQ, AhRcomp and POPs/PCBs/pesticides observed might be due to a higher concentration of compounds having antagonistic activity on AhR transactivity.

The biomagnification of PCBs in the aquatic food-chain is congener-specific. For most marine mammals, non-DL-PCBs are observed more resistant to biodegradation than DL-PCBs 60 and a previous study reported that the concentration of CB77, CB126 and CB169 showed no obvious biomagnification although the concentration of non-DL-PCBs such as CB138 increased from plankton to piscivores to herring gulls 61. The lack of biomagnification of CB77 was attributed to its rapid elimination by aquatic species 62 and the CB105 and CB118 are more easily metabolized congeners than non-DL-PCBs such as CB153 63. Owing to this selective biotransformation during the bioaccumulation in the food chain 10, the concentration of some DL-PCBs might be reduced, whereas non-DL-PCBs accumulate, causing the variation of the composition of bioaccumulated PCBs.

In overall, the serum AhR-TEQ/AhRcomp negatively correlated to age and n-3/n-6 ratio, being in accordance to the frequency of marine food intake and POPs bioaccumulation over time. For the separate districts and genders a scattered correlation of AhR-TEQ/AhRcomp and lifestyle factors was observed. This non-consistent pattern might be explained by relative low statistical power for the separate gender and/or districts and needs further confirmation.

The inverse correlation of AhR-TEQ to seafood intake given by the n-3/n-6 ratio, suggests that the AhR transcriptional activity might follow the intake of n-6 fatty acids possibly with the origin from non-marine diet being supported by our previous report (Bonefeld-Jorgensen and Long, submitted). Moreover, it was reported that the important source of DL-compounds for the population of the western world is dairy food intake 646566. In the present study, we observed that the consumption of dairy food was higher for the Nuuk and Sisimiut participants than that of Qaanaaq and a positive correlation between dairy food intake and serum AhR-TEQ level was observed for the combined Nuuk and Sisimiut data and the combined Nuuk, Sisimiut and Qaanaaq data, respectively. Being more westernized, the import of western food and intake of milk products was reported to be higher for the subjects living in Nuuk and Sisimiut, while the intake of local Greenlandic products was relatively higher in Qaanaaq 10. Hence, the dairy food might be an important source of DL-compounds for the Inuits living in bigger cities such as Nuuk and Sisimiut, relating to the higher AhR-TEQ level observed for Nuuk and Sisimiut compared to Qaanaaq. Moreover, we observed that POPs increase significantly with age for Inuits in this study as reported 10 as well as for Inuits and Europeans in an earlier study 67. Evaluation of our previous data 27 showed that in Europeans the AhR-TEQ positively correlated to age (r = 0.12, p = 0.05), whereas for Inuits negative correlation was observed (r = -0.3, p = 0.01), suggesting different POP profiles resulted in different outcome on the AhR transcriptional activity.

Although the correlations found in the present study were weak, the chance finding may be excluded because the significant correlations were higher than 5%.

In the present study, the AhR transcriptional activation of serum extract was measured as an integrated net AhR-TEQ value without providing information on the levels of individual congeners. This bioassay responds to environmental contaminants acting through the AhR-pathway and allows the detection of chemicals that are not included in the chemical analytical method based on the standard TEF scheme. However, as discussed previously 27, upon serum extraction and clean-up the polycyclic aromatic hydrocarbons (PAHs) and other plant dietary AhR agonists are not expected to contribute to our determined AhR transcriptional activity (personal communication, Pierre Dumas, Quebec, Canada). The CALUX bioassay including AhR transcription activation assay takes into account all possible interactions (synergistic, additive and/or antagonistic interaction) between congeners while chemical analytical method like GC-MS focuses on a selected numbers of compounds (i.e. those documented to produce AhR-dependent toxicity) and only assumes the additive interaction of known detectable compounds. Therefore, the AhR-TEQ value derived from CALUX bioassays (AhR transcription activation bioassay) and chemical methods (WHO-TEQ) can not be expected to be equal. In most of the cases, higher AhR-TEQ value are usually obtained with AhR-CALUX bioassays compared to the calculated WHO-TEQ derived from chemical method 1868. However, the AhR-TEQ and WHO-TEQ values derived from AhR transcriptional activation CALUX bioassays and chemical analyses, respectively, are correlated 68 and these two methods are complimentary where the AhR transcription activation CALUX bioassay provides an overall biological response of the mixture, whereas the chemical analysis provides the concentration of specific compounds. Thus the key application of AhR transcriptional activation CALUX bioassay is screening and prioritization of samples 28 and is a valuable tool for comparison of different level of AhR transcriptional activity including DL-activity across various population.

5. Conclusion

The POPs and serum AhR transcriptional activity differed among Greenlandic districts, suggesting that the difference of serum AhR transcriptional activity depends on the composition of bioaccumulated POPs. The observed inverse correlation of POPs and AhR-TEQ or AhRcomp data suggests the presence of compounds with antagonistic impact on the AhR signalling pathway, probably due to the selective PCB bioaccumulation in the food chain. Further study is needed to elucidate this preliminary conclusion.

Abbreviations

AhR aryl hydrocarbon receptor

AhRag agonistic AhR activity

AhRcomp competitive AhR activity

CALUX Chemical activated luciferase gene expression

DDT 2, 2-bis (p-chlorophenyl)-1, 1, 1-trichloroethane

DL dioxin-like

GC-MS gas chromatography – mass spectrometry

HCB hexachlorobenzene

β-HCH Hexachlorocyclohexane

PAHs polycyclic aromatic hydrocarbons

PCDDs/PCDFs polychlorinated dibenzo-p-dioxins/furans

PCBs polychlorinated biphenyls

POPs persistent organic pollutants

TCDD 2,3,7,8-tetrachlorodibenzo-p-dioxin

TEFs Toxic Equivalency Factors

TEQs TCDD toxic equivalents

Competing interests

The author(s) declare that they have no competing interests.

Authors' contributions

ML and EBJ were responsible for design of the AhR transcriptional activity study and preparation of the manuscript; ML performed the mechanistic work and data evaluation. BD has provided the epidemiological data concerning POPs, questionnaires and fatty acids. All authors have read and approved the final manuscript.

Acknowledgements

We thank technical assistant Birgitte S Andersen and Inger Sørensen for their excellent skills in the laboratory work and all CMT group members for scientific support. We thank Jens C Hansen for comments on the manuscript and Henning S Pedersen for sampling. This study was supported by DANCEA, The Danish Environmental Ministry as a part of the "AMAP Human Health Effects Monitoring programme", and by the EU commission (INUENDO project).

 <p>Toxic equivalency factors (TEFs) for PCBs, PCDDs, PCDFs for humans and wildlife</p> Van den Berg M Birnbaum L Bosveld AT Brunstrom B Cook P Feeley M Giesy JP Hanberg A Hasegawa R Kennedy SW Kubiak T Larsen JC van Leeuwen FX Liem AK Nolt C Peterson RE Poellinger L Safe S Schrenk D Tillitt D Tysklind M Younes M Waern F Zacharewski T Environ Health Perspect 1998 106 12 775 792 1533232 9831538 10.2307/3434121 <p>Polychlorinated biphenyls (PCBs): environmental impact, biochemical and toxic responses, and implications for risk assessment</p> Safe SH Crit Rev Toxicol 1994 24 2 87 149 8037844 <p>Chlorobenzenes, chlorinated pesticides, coplanar chlorobiphenyls and other organochlorine compounds in Greenland biota</p> Vorkamp K Riget F Glasius M Pecseli M Lebeuf M Muir D Sci Total Environ 2004 331 1-3 157 175 10.1016/j.scitotenv.2004.03.027 15325147 <p>Arctic contaminants: sources, occurrence and pathways</p> Barrie LA Gregor D Hargrave B Lake R Muir D Shearer R Tracey B Bidleman T Sci Total Environ 1992 122 1-2 1 74 10.1016/0048-9697(92)90245-N 1514103 <p>High human plasma levels of organochlorine compounds in Greenland. Regional differences and lifestyle effects</p> Deutch B Hansen JC Dan Med Bull 2000 47 2 132 137 10822803 <p>Circumpolar maternal blood contaminant survey, 1994-1997 organochlorine compounds</p> Van Oostdam JC Dewailly E Gilman A Hansen JC Odland JO Chashchin V Berner J Butler-Walker J Lagerkvist BJ Olafsdottir K Soininen L Bjerregard P Klopov V Weber JP Sci Total Environ 2004 330 1-3 55 70 10.1016/j.scitotenv.2004.02.028 15325158 <p>Toxicological properties of persistent organic pollutants and related health effects of concern for the arctic populations</p> Bonefeld Jorgensen EC Ayotte P AMAP Assessment 2002: Human Health in the Arctic 2003 chapter 6 57 74 <p>Dietary composition in Greenland 2000, plasma fatty acids and persistent organic pollutants</p> Deutch B Pedersen HS Hansen JC Sci Total Environ 2004 331 1-3 177 188 10.1016/j.scitotenv.2004.03.028 15325148 <p>Dietary composition and contaminants in north Greenland, in the 1970s and 2004</p> Deutch B Dyerberg J Pedersen HS Asmund G Moller P Hansen JC Sci Total Environ 2006 370 372 381 10.1016/j.scitotenv.2006.07.015 16904734 <p>Contaminants, diet, plasma fatty acids and smoking in Greenland 1999-2005</p> Deutch B Pedersen HS Asmund G Hansen JC Sci Total Environ 2007 372 2-3 486 496 10.1016/j.scitotenv.2006.10.043 17157358 <p>Inuit foods and diet: a preliminary assessment of benefits and risks</p> Kinloch D Kuhnlein H Muir DC Sci Total Environ 1992 122 1-2 247 278 10.1016/0048-9697(92)90249-R 1514105 <p>Concentration of organochlorines in human brain, liver, and adipose tissue autopsy samples from Greenland</p> Dewailly E Mulvad G Pedersen HS Ayotte P Demers A Weber JP Hansen JC Environ Health Perspect 1999 107 10 823 828 1566611 10504150 10.2307/3454581 <p>Dioxin revisited: developments since the 1997 IARC classification of dioxin as a human carcinogen</p> Steenland K Bertazzi P Baccarelli A Kogevinas M Environ Health Perspect 2004 112 13 1265 1268 1247514 15345337 <p>Workshop on perinatal exposure to dioxin-like compounds. I. Summary</p> Lindstrom G Hooper K Petreas M Stephens R Gilman A Environ Health Perspect 1995 103 Suppl 2 135 142 1518837 7614935 10.2307/3432459 <p>Aryl hydrocarbon receptor-mediated signal transduction</p> Rowlands JC Gustafsson JA Crit Rev Toxicol 1997 27 2 109 134 9099515 <p>Cellular and molecular biology of aryl hydrocarbon (Ah) receptor-mediated gene expression</p> Safe S Krishnan V Arch Toxicol Suppl 1995 17 99 115 7786196 <p>The 2005 World Health Organization reevaluation of human and Mammalian toxic equivalency factors for dioxins and dioxin-like compounds</p> Van den Berg M Birnbaum LS Denison M De Vito M Farland W Feeley M Fiedler H Hakansson H Hanberg A Haws L Rose M Safe S Schrenk D Tohyama C Tritscher A Tuomisto J Tysklind M Walker N Peterson RE Toxicol Sci 2006 93 2 223 241 10.1093/toxsci/kfl055 16829543 <p>Trace contamination with dioxin-like chemicals: evaluation of bioassay-based TEQ determination for hazard assessement and regulatory responses</p> Van Overmeire I Clark, G.C., Brown, D.J.,Chu, M.D.,Cooke, W. M., Denison, M.S., Baeyene, W., Srebrnik S.,Goeyens, L. Environmental Science & Policy 2001 4 6 345 357 10.1016/S1462-9011(01)00037-5 <p>Applicability of the CALUX bioassay for screening of dioxin levels in human milk samples</p> Laier P Cederberg,T, Larsen, J.C., Vinggaard, A.M. Food Additives and Contaminants 2003 20 6 583 595 10.1080/0265203031000107820 <p>Species-specific antagonism of Ah receptor action by 2,2',5,5'-tetrachloro- and 2,2',3,3'4,4'-hexachlorobiphenyl</p> Aarts JM Denison MS Cox MA Schalk MA Garrison PM Tullis K de Haan LH Brouwer A Eur J Pharmacol 1995 293 4 463 474 10.1016/S0014-2999(95)80105-7 8748700 <p>Effects of currently used pesticides in the AhR-CALUX assay: comparison between the human TV101L and the rat H4IIE cell line</p> Long M Laier P Vinggaard AM Andersen HR Lynggaard J Bonefeld-Jorgensen EC Toxicology 2003 194 1-2 77 93 10.1016/j.tox.2003.08.001 14636698 <p>Bioanalytical screening methods for dioxins and dioxin-like compounds a review of bioassay/biomarker technology</p> Behnisch PA Hosoe K Sakai S Environ Int 2001 27 5 413 439 10.1016/S0160-4120(01)00028-9 11757855 <p>Human risk assessment and TEFs</p> van den Berg M Peterson RE Schrenk D Food Addit Contam 2000 17 4 347 358 10.1080/026520300283414 10912248 <p>Species-specific recombinant cell lines as bioassay systems for the detection of 2,3,7,8-tetrachlorodibenzo-p-dioxin-like chemicals</p> Garrison PM Tullis K Aarts JM Brouwer A Giesy JP Denison MS Fundam Appl Toxicol 1996 30 2 194 203 10.1006/faat.1996.0056 8812265 <p>Chemical-activated luciferase gene expression (CALUX): a novel in vitro bioassay for Ah receptor active compounds in sediments and pore water</p> Murk AJ Legler J Denison MS Giesy JP van de Guchte C Brouwer A Fundam Appl Toxicol 1996 33 1 149 160 10.1006/faat.1996.0152 8812260 <p>Application of the chemical-activated luciferase expression (CALUX) bioassay for quantification of dioxin-like compound in small samples of human milk and blood plasma</p> Aarts JMMJG Cenjin PH Blankvoort BMG Murk A Brouwer A Bovee TFH Traag WA Hoogenboom LAP Patandin S Weisglas-Kuperus N Sauer PJJ Dnison MS Organohal Cmpds 1996 27 285 290 <p>Dioxin-like activities in serum across European and Inuit populations</p> Long M Andersen BS Lindh CH Hagmar L Giwercman A Manicardi GC Bizzaro D Spano M Toft G Pedersen HS Zvyezday V Bonde JP Bonefeld-Jorgensen EC Environ Health 2006 5 14 1501006 16725033 10.1186/1476-069X-5-14 <p>Chemically activated luciferase gene expression (CALUX) cell bioassay analysis for the estimation of dioxin-like activity: critical parameters of the CALUX procedure that impact assay results</p> Windal I Denison MS Birnbaum LS Van Wouwe N Baeyens W Goeyens L Environ Sci Technol 2005 39 19 7357 7364 10.1021/es0504993 16245802 <p>Functional aspects of developmental toxicity of polyhalogenated aromatic hydrocarbons in experimental animals and human infants</p> Brouwer A Ahlborg UG Van den Berg M Birnbaum LS Boersma ER Bosveld B Denison MS Gray LE Hagmar L Holene E Eur J Pharmacol 1995 293 1 1 40 10.1016/0014-2999(95)90111-6 7545581 <p>Arctic Monitoring and Assessment Programme </p> AMAP http://wwwamapno/ <p>Organochlorine levels in maternal and umbilical cord blood plasma in Arctic Canada</p> Butler Walker J Seddon L McMullen E Houseman J Tofflemire K Corriveau A Weber JP Mills C Smith S Van Oostdam J Sci Total Environ 2003 302 1-3 27 52 10.1016/S0048-9697(02)00319-4 12526896 <p>Smoking as a determinant of high organochlorine levels in Greenland</p> Deutch B Pedersen HS Jorgensen EC Hansen JC Arch Environ Health 2003 58 1 30 36 10.3200/AEOH.58.1.30-36 12747516 <p>Adipose tissue fatty acids as biomarkers of dietary exposure in Danish men and women</p> Tjonneland A Overvad K Thorling E Ewertz M Am J Clin Nutr 1993 57 5 629 633 8480677 <p>Development and modification of a recombinant cell bioassay to directly detect halogenated and polycyclic aromatic hydrocarbons in serum</p> Ziccardi MH Gardner IA Denison MS Toxicol Sci 2000 54 1 183 193 10.1093/toxsci/54.1.183 10746945 <p>Effect of pesticides on estrogen receptor transactivation in vitro: a comparison of stable transfected MVLN and transient transfected MCF-7 cells</p> Bonefeld-Jorgensen EC Grunfeld HT Gjermandsen IM Mol Cell Endocrinol 2005 244 1-2 20 30 10.1016/j.mce.2005.01.017 16219411 <p>Persistent organochlorine pollutants in human serum of 50-65 years old women in the Flanders Environmental and Health Study (FLEHS). Part 2: Correlations among PCBs, PCDD/PCDFs and the use of predictive markers</p> Covaci A Koppen G Van Cleuvenbergen R Schepens P Winneke G van Larebeke N Nelen V Vlietinck R Schoeters G Chemosphere 2002 48 8 827 832 10.1016/S0045-6535(02)00112-1 12222776 <p>Comparison of chemical-activated luciferase gene expression bioassay and gas chromatography for PCB determination in human serum and follicular fluid</p> Pauwels A Cenijn PH Schepens PJ Brouwer A Environ Health Perspect 2000 108 6 553 557 1638137 10856030 10.2307/3454618 <p>Dioxin-Like TEQ of women from the Seveso, Italy area by ID-HRGC/HRMS and CALUX</p> Warner M Eskenazi B Patterson DG Clark G Turner WE Bonsignore L Mocarelli P Gerthoux PM J Expo Anal Environ Epidemiol 2004 <p>Biomarker measurements in a coastal fish-eating population environmentally exposed to organochlorines</p> Ayotte P Dewailly E Lambert GH Perkins SL Poon R Feeley M Larochelle C Pereg D Environ Health Perspect 2005 113 10 1318 1324 1281273 16203240 <p>Determinants of serum polychlorinated biphenyls and organochlorine pesticides measured in women from the child health and development study cohort, 1963-1967</p> James RA Hertz-Picciotto I Willman E Keller JA Charles MJ Environ Health Perspect 2002 110 7 617 624 1240905 12117636 <p>Levels and spatial and temporal trends of contaminants in Greenland biota: an updated review</p> Riget F Dietz R Vorkamp K Johansen P Muir D Sci Total Environ 2004 331 1-3 29 52 10.1016/j.scitotenv.2004.03.022 15325140 <p>Human exposure to contaminants in the traditional Greenland diet</p> Johansen P Muir D Asmund G Riget F Sci Total Environ 2004 331 1-3 189 206 10.1016/j.scitotenv.2004.03.029 15325149 <p>PCBs and dioxin-like compounds in plasma of adult Inuit living in Nunavik (Arctic Quebec)</p> Ayotte P Dewailly E Ryan JJ Bruneau S Lebel G Chemosphere 1997 34 5-7 1459 1468 10.1016/S0045-6535(97)00442-6 9134679 <p>Sampling and measurement of PCDD/PCDF and non-ortho PCB in Arctic air at Ny-Ålesund, Spitzbergen</p> Schlabach M Biseth A Gundersen H Organohal Cmpds 1996 28 325 239 <p>Mixture effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin and polychlorinated biphenyl congeners in rats</p> Chu I Lecavalier P Hakansson H Yagminas A Valli VE Poon P Feeley M Chemosphere 2001 43 4-7 807 814 10.1016/S0045-6535(00)00437-9 11372869 <p>Interaction between halogenated aromatic compounds in the Ah receptor signal transduction pathway</p> Chen G Bunce NJ Environ Toxicol 2004 19 5 480 489 10.1002/tox.20053 15352264 <p>Aroclor 1254 as a 2,3,7,8-tetrachlorodibenzo-p-dioxin antagonist: effects on enzyme induction and immunotoxicity</p> Bannister R Davis D Zacharewski T Tizard I Safe S Toxicology 1987 46 1 29 42 10.1016/0300-483X(87)90135-1 3116725 <p>Antagonism of aryl hydrocarbon receptor-dependent induction of CYP1A1 and inhibition of IgM expression by di-ortho-substituted polychlorinated biphenyls</p> Suh J Kang JS Yang KH Kaminski NE Toxicol Appl Pharmacol 2003 187 1 11 21 10.1016/S0041-008X(02)00040-6 12628580 <p>Study of the interference problems of dioxin-like chemicals with the bio-analytical method CALUX </p> Schroijen C Windal I Goeyens L Baeyens W Talanta 2004 63 1261 1268 10.1016/j.talanta.2004.05.036 <p>Differential regulation of cytochrome P450 1A1 and 1B1 by a combination of dioxin and pesticides in the breast tumor cell line MCF-7</p> Coumoul X Diry M Robillot C Barouki R Cancer Res 2001 61 10 3942 3948 11358810 <p>Effect of toxaphene on estrogen receptor functions in human breast cancer cells</p> Bonefeld Jorgensen EC Autrup H Hansen JC Carcinogenesis 1997 18 8 1651 1654 10.1093/carcin/18.8.1651 9276643 <p>The Human Health Effect Programme in Greenland, a review</p> Bonefeld-Jorgensen EC Sci Total Environ 2004 331 1-3 215 231 10.1016/j.scitotenv.2004.03.030 15325151 <p>Estrogenic action of DDT and its analogs</p> Welch RM Levin W Conney AH Toxicol Appl Pharmacol 1969 14 2 358 367 10.1016/0041-008X(69)90117-3 5772860 <p>The effects of chlordane exposure during pre- and postnatal periods at environmentally relevant levels on sex steroid-mediated behaviors and functions in the rat</p> Cassidy RA Vorhees CV Minnema DJ Hastings L Toxicol Appl Pharmacol 1994 126 2 326 337 10.1006/taap.1994.1123 8209386 <p>Effect of highly bioaccumulated polychlorinated biphenyl congeners on estrogen and androgen receptor activity</p> Bonefeld-Jorgensen EC Andersen HR Rasmussen TH Vinggaard AM Toxicology 2001 158 3 141 153 10.1016/S0300-483X(00)00368-1 11275356 <p>Assessment of xenoestrogenic exposure by a biomarker approach: application of the E-Screen bioassay to determine estrogenic response of serum extracts</p> Rasmussen TH Nielsen F Andersen HR Nielsen JB Weihe P Grandjean P Environ Health 2003 2 1 12 270076 14613489 10.1186/1476-069X-2-12 <p>Effects of o,p'-DDT on the 2,3,7,8-tetrachlorodibenzo-p-dioxin-inducible CYP1A1 expression in murine Hepa-1c1c7 cells</p> Jeong HG Kim JY Food Chem Toxicol 2002 40 11 1685 1692 10.1016/S0278-6915(02)00099-6 12176094 <p>Endocrine disrupting potentials of bisphenol A, bisphenol A dimethacrylate, 4-n-nonylphenol and 4-n-octylphenol in vitro-new data and a brief review</p> Bonefeld-Jorgensen EC Long M M.V. H Vinggaard AM Environ Health Perspect 2007 in press http://www.ehponline.org/docs/admin/newest.html <p>Synergistic effect of 2,2',4,4',5,5'-hexachlorobiphenyl and 2,3,7,8-tetrachlorodibenzo-p-dioxin on hepatic porphyrin levels in the rat</p> van Birgelen AP Fase KM van der Kolk J Poiger H Brouwer A Seinen W van den Berg M Environ Health Perspect 1996 104 5 550 557 1469354 8743444 10.2307/3432997 <p>Concentration-dependent changes of PCB patterns in fish-eating mammals: structural evidence for induction of cytochrome P450</p> Boon JP van der Meer J Allchin CR Law RJ Klungsoyr J Leonards PE Spliid H Storr-Hansen E McKenzie C Wells DE Arch Environ Contam Toxicol 1997 33 3 298 311 10.1007/s002449900257 9353208 <p>Concise International Chemical Assessment Document 55: Polychlorinated biphenyls: human health aspects </p> IPCS http://www.inchem.org/documents/cicads/cicads/cicad55.htm 2003 <p>The distribution of 42 PCBs, including three coplanar congeners, in the food web of the western basin of Lake Erie</p> Koslowski SE Metcalfe CD Lazar R Haffner GD journal of Great Lakes Research 1994 20 260 270 <p>Plasma concentrations of polychlorinated biphenyls and the risk of breast cancer: a congener-specific analysis</p> Demers A Ayotte P Brisson J Dodin S Robert J Dewailly E Am J Epidemiol 2002 155 7 629 635 10.1093/aje/155.7.629 11914190 <p>Dioxins, dioxin-like PCBs and non-dioxin-like PCBs in foodstuffs: occurrence and dietary intake in The Netherlands</p> Baars AJ Bakker MI Baumann RA Boon PE Freijer JI Hoogenboom LA Hoogerbrugge R van Klaveren JD Liem AK Traag WA de Vries J Toxicol Lett 2004 151 1 51 61 10.1016/j.toxlet.2004.01.028 15177640 <p>Human exposure to dioxins through the diet in Catalonia, Spain: carcinogenic and non-carcinogenic risk</p> Llobet JM Domingo JL Bocio A Casas C Teixido A Muller L Chemosphere 2003 50 9 1193 1200 10.1016/S0045-6535(02)00630-6 12547333 <p>Human exposure to dioxins through diet in Italy</p> Taioli E Marabelli R Scortichini G Migliorati G Pedotti P Cigliano A Caporale V Chemosphere 2005 61 11 1672 1676 10.1016/j.chemosphere.2005.04.032 15939454 <p>Inter-population variations in concentrations, determinants of and correlations between 2,2',4,4',5,5'-hexachlorobiphenyl (CB-153) and 1,1-dichloro-2,2-bis (p-chlorophenyl)-ethylene (p,p'-DDE): a cross-sectional study of 3161 men and women from Inuit and European populations</p> Jonsson BA Rylander L Lindh C Rignell-Hydbom A Giwercman A Toft G Pedersen HS Ludwicki JK Goralczyk K Zvyezday V Spano M Bizzaro D Bonefeld-Jorgensen EC Manicardi GC Bonde JP Hagmar L Environ Health 2005 4 27 1308838 16283941 10.1186/1476-069X-4-27 <p>Validation of the CALUX bioassay for PCDD/F analyses in human blood plasma and comparison with GC-HRMS </p> Van Wouwe N Windal I Vanderperren H