The increasing load of the environment by man-made pollutants is of concern for the human health. Due to long-range transport by atmospheric and oceanic currents 12 the human exposure is not limited to individuals living close to the sources of the contaminants. The lipophilic persistent organic pollutants (POPs) includes polychlorinated dibenzo-p-dioxins/furans (PCDDs/PCDFs), polychlorinated biphenyls (PCBs) and certain organochlor pesticide residues e.g. 2,2-bis(p-chlorophenyl)-1,1,1-trichloroethane (DDT), its metabolite 1,1-dichloro-2,2-bis (p-chlorophenyl)-ethylene (p,p'-DDE), toxaphenes and chlordanes. Due to their high lipophilicity and resistance to biodegradation POPs are biomagnified through the food chain and found in fatty tissues at high concentrations in predator fish and birds, seals, whales and polar bears 3. Greenlandic Inuit display high body burden of POPs 4, that significantly correlate with age, smoking and the level of n-3 polyunsaturated fatty acids in plasma, being strong indicators of the main source of POP contaminants in their traditional marine food 56789.

In some Greenlandic districts the PCB body burden levels exceed Health Canada guidelines of concern (5 – 100 μl/L plasma) 10. Also within Greenland strong regional differences are observed with the highest contaminant levels found in Inuit at the East coast 10, where also higher levels of PCBs and DDT are found in marine species and birds compared to the West coast 1112. Some districts and settlements such as Qaanaaq (North West) still primarily rely on traditional foods, whereas the diet in the cities Nuuk and Sisimiut (South West) are more westernized 1013.

During the last two decades, a number of POPs have been identified and characterized as being estrogenic, antiestrogenic and/or antiandrogenic 1415161718, and to have a multitude of potential health effects on wildlife and humans, including immunotoxicity, carcinogenicity, and adverse effects on reproductive, neurobehavioral, and endocrine functions 192021222324. Recently also endocrine-related human health effects of POPs on child development were reported at the individual or population level 252627. However, the toxicological assessment of POPs on humans is complicated. Not only is the analytical chemical approach for the detection of all xenobiotics practically impossible especially on large-scale surveys but additive enhancement of hormone actions has been reported in vitro for xenoestrogen mixtures 2829 and recently in vivo for antiandrogens 30. Therefore, the assessment of the integrated biological effect of the actual chemical mixture in human blood is important and ex vivo cell systems have recently been introduced to enable the assessment of the integrated level of xenobiotic transactivity in human adipose tissue 203132 or in human serum 3334353637. We have previously validated the SPE-HPLC serum POP extraction method and it was demonstrated that the ER- and AR-CALUX assays respond to a wide range of different compounds including PCBs and pesticides 1415 as well as to PCB-spiked serum extracts 35.

The aims were 1) to determine the integrated xenohormone bioactivities as an exposure marker of the actual lipophilic serum POP mixture measuring the effect on estrogen (ER) and androgen receptor (AR) transactivity in cerum of citizens from different Greenlandic districts and 2) to evaluate associations of the xenohormone transactivities to serum POP markers (14 PCBs and 10 pesticides) and lifestyle characteristics.

In the present study we determined the actual xenohormone transactivity as a biomarker of serum POP exposure and evaluated whether the transactivity was associated to serum POP concentrations and the lifestyle characteristics across the Greenlandic districts Nuuk, Sisimiut and Qaanaaq. For the age and/or n-3/n-6 adjusted data we found an inverse correlation between xenoestrogenic and xenoandrogenic transactivities for combined female and combined male, respectively.

In general, serum POP extracts, free of endogenous hormones, showed a trend to decrease the ER transactivity (XER) and a low trend to agonize the AR transactivity (XAR) for both genders. In order to assure that the effect of the actual serum POP mixture on the receptor activation does reflect the biological activity in serum and the in vivo interaction between endogenous hormones and the POPs, we did evaluate the effect of the serum POP extracts in the presence of relevant concentrations of high affinity ligands (E2 and R1881) for the receptors to mimic the in vivo potential of the serum samples (XERcomp and XARcomp).

In the single districts XERcomp and XARcomp transactivities were predominantly antagonized except for serum extracts of the older Nuuk men where 50% elicited further increased XARcomp transactivities. No significant differences in the XER and the XAR were observed between men and women except the XAR activity of Sisimiut men being higher than Sisimiut women supporting that the endogenous hormones were effectively separated from the serum POP fractions.

Because of differences between the genders in lifestyle characteristics and POP levels and to improve the statistical power, we evaluated the combined data of each gender across the districts.

In the present study the serum samples of men from Nuuk contained a higher level of POPs due to their higher average age and higher intake of seafood and seabird 7. However, in accordance with previous studies, Qaanaaq inhabitants, having a high intake of marine and traditional diet, had in general significantly higher levels of POPs compared to Nuuk women and Sisimiut inhabitants, eating a more westernized diet. For all three districts the serum POP levels of men were significantly higher than for women, reflecting the higher intake of traditional food by men 7. Moreover, despite a high intercorrelation between POPs, the profile differed between districts and genders reflecting different exposure pattern.

In Arctic populations higher serum POP concentrations is determined by age and intake of marine food as reflected by an increased n-3/n-6 fatty acid ratio 842, and a higher level of POPs was reported for smokers 79. We observed positive associations between the xenohormone transactivities and age, n-3/n-6 and smoker years for the combined male data whereas the xenoestrogenic activities were negatively associated to n-3/n-6 for the combined women. Thus, the determinants of the serum POP concentration seem also to be predictors of the xenohormone transactivities supporting the use of xenohormone transactivities as an integrated biomarker of POP exposure and lifestyle characteristics.

For combined men the positive correlation between the XERcomp transactivities and the POPs disappeared upon adjustment for age. POP concentrations increase by age, and age was found to be a determinant of the XERcomp transactivities for men. Thus, the observed association between XERcomp and the unadjusted POP data might be a reflection of the broad range of age for the included men and the correlation between XERcomp and age.

The XERcomp transactivities for the combined women correlated inversely to both non-adjusted and age-adjusted POPs. We found in a previous study 33, evaluating the xenoestrogenic serum transactivity of male study groups from Europe and Greenland, a negative correlation between the ER transactivity and the two POP proxy markers PCB153 and p,p'-DDE for Inuit.

Dioxins are known to exert antiestrogenic actions 4344. In the same study population as used in this study, the serum dioxin-like transactivity mediated via the aryl hydrocarbon receptor (AhR) was determined 45. Interestingly, the xenoestrogenic transactivities correlated negatively to the AhR transactivities for the combined women supporting the presence of antiestrogenic POPs in the serum POP mixture. In support, although using another approach it was recently reported that high levels of PCBs in Slovakia male serum samples were associated with a decreased ER mediated activity and increased AhR mediated activity 41.

Data on the xenoestrogenic activity in human samples are still scarce. Few studies using a different setup of measurement of xenoestrogenic transactivities in adipose tissue from Spanish women and serum samples from pregnant Danish and Faroese women have been reported, although, in contrast to our studies no correlation between POPs and MCF-7 proliferation were found 32203134. However, recent data showed, for non-pregnant female greenhouse workers in Denmark that the E2-induced MCF-7 proliferation response was reduced, indicating an antiestrogenic effect of the serum extract containing major xenoestrogens but without pharmaceutical and endogenously produced estrogens 46. We believe that the high impact of POP burden in the Inuit is responsible for the antagonized ER function in line with earlier in vitro studies 1534.

In the present study only a small fraction of the serum POP extracts agonized the XAR transactivities. Numerous POPs have been shown to be antiandrogenic in vivo and in vitro 174714184815495051. However, also androgenic effects have been found in the environment, e.g. craft mill effluent from a river in Florida contained a chemical mixture that induced AR-dependent gene expression and masculinized female fish in vitro 52.

The in vivo mimicking XARcomp transactivities were predominantly antagonized supporting the in vivo and in vitro antiandrogenic effects of the POPs. However, for 50% of the older Nuuk men a further increased XARcomp transactivity was observed. Some AhR agonists elicit antiandrogenic effects although the exact mechanisms are not clear 535455. In the same study population as used in this study, the 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)-induced AhR transactivities (AhRcomp) were significantly lower for Nuuk men compared to men from Sisimiut and Qaanaaq 45. Furthermore, 32% of the serum samples of Nuuk men inhibited AhRcomp transactivity compared to 4% and 2% of the serum samples of Sisimiut and Qaanaaq men, respectively 45. Thus, the serum extracts of Nuuk men seems to include some POPs further increasing the XARcomp transactivity but inhibiting the AhRcomp transactivity. Whether these POPs are related to the higher age of Nuuk men or their higher intake of seafood and seabird can at this point not be determined.

Taking the broad age range of the men across the districts into account the combined male XARcomp data correlated negatively to the POPs upon adjustment for age. Because of the higher age of Nuuk men we also analyzed the combined data of Sisimiut and Qaanaaq men separately, and significant negative correlations to the POPs were observed both before and after adjustment for age. In support to the present results, we previously observed negative correlations between XARcomp and the POP proxy marker p,p'-DDE for combined European study groups 36.

The data showed correlations between POPs and female xenoestrogenic and male xenoandrogenic transactivities. We cannot explain this gender pattern but have the following suggestions: The serum POP concentration was approximately 3 times higher in male serum compared to female serum mainly because of a different diet pattern, men eating more traditional and women more westernized 7, resulting in a different compositions of the serum POP mixture between the two sexes.

We believe that the presented data reflects the effect of POPs on the sex hormone receptors as supported by the following: the observed correlations between xenohormone transactivities and the POPs were all observed in the in vivo mimic by the presence of a high affinity receptor ligand (E2 for ER and R1881 for AR). No correlations were observed for the serum POP extracts alone and furthermore, the observed correlations were negative. Thus, it does not seem plausible that the correlations between XERcomp/XARcomp and the age-adjusted POPs result from remnant endogenous hormones in the serum POP fraction. However, we cannot assess whether the correlations in the present study are only by chance findings. 25 out of 276 correlations (<10%) showed statistically significance, whereas the expected number of significant findings under the null hypothesis of no difference would be 14 out of 276 correlations (5%). We did observe fewer significant findings for the xenoandrogenic transactivities. This could be a result of the similar coefficients of variation within and between days for the AR-CALUX assay making the assay less robust than the ER assay.

Interestingly, we found higher xenoandrogenic/xenoestrogenic ratio for the male Inuit compared to the daily serum controls of Danish men (see section 2.3) as well as compared to European male in another study 36. At this point it is not possible to assess whether this change in xenoandrogenicity can have any impact in vivo on the hormone homeostasis. It was proposed, that exposure to a mixture of chemicals with antiandrogenic or estrogenic properties may affect the androgen-estrogen ratio and thereby influence the risk of cryptorchidism in Danish and Finnish boys 56. Whether a lower rate of cryptorchidism is found in Inuit awaits further research. We did previously observe negative correlations for xenoestrogenic and dioxin-like transactivity to sperm chromatin integrity and DNA damage for Inuit and positive correlations between xenoandrogenic and dioxin-like transactivity and sperm chromatin integrity and DNA damage for Europeans 5758. Further studies are needed to elucidate the possible association between POP related xenohormone transactivity and human health risk. Furthermore, other types of lipophilic endocrine disrupters in serum like the brominated flame retardants (BFRs) and perfluorooctanesulfonated (PFOS) affect the receptor function and should in future studies be taken into consideration.

Whether our ex vivo data mimic the cellular and molecular effects in vivo awaits additional studies. The presented data gives the final integrated net effect on the given receptor alone and other factors such as receptor and/or co-factor interactions might precede the final data. Our biomarker approach gives the possibility to estimate the combined impact of complex mixtures on health risks mediated via sex hormone receptors.

Determinants of serum POP concentration are also predictors of the xenohormone transactivities and can be used as an integrated biomarker of POP exposure and lifestyle characteristics. The actual serum POP mixture antagonize age adjusted sex hormone actions as supported by the inverse correlations to the xenoestrogenic and the xenoandrogenic transactivities for the combined female and the combined male Inuit, respectively.

The data of the presented manuscript is the first of its kind ever published for Inuit having very high levels of POPs. The emerging changes in diet in Greenland and thus the observed district differences in POP concentrations as well as mixture profile will be of great importance for the future elucidation of how mixtures of different POP levels and mixtures among different geographical population might affect the risk of health.

The authors declare that they have no competing interests.

ECBJ was responsible for the design of the study. TK, MG and ECBJ drafted the manuscript and were responsible for data evaluation and statistical analyses. TK carried out the AR-CALUX analyses. MG carried out the ER-CALUX analyses. PSH carried out the SPE-HPLC fractionation of the serum samples and commented on the manuscript. BD evaluated the POP data and questionnaires concerning lifestyle characteristics and established the database. All authors read and approved the final manuscript.