Antagonistic activity towards the androgen receptor independent from natural sex hormones in human milk samples from the Norwegian HUMIS cohort

In this paper, we investigated the possible presence of endocrine disrupting chemicals (EDCs) based on measuring the total estrogenic and androgenic activity in human milk samples. We used specific bioassays for analysis of the endocrine activity of estrogens and estrogen-like EDCs and androgens and androgen-like EDCs and developed a separation method to evaluate the contribution from natural hormones in comparison to that of EDCs to total endocrine activities. We extracted ten random samples originating from the Norwegian HUMIS biobank of human milk and analyzed their agonistic or antagonistic activity using the ERα- and AR CALUX® bioassays. The study showed antagonistic activity towards the androgen receptor in 8 out of 10 of the assessed human milk samples, while 2 out of 10 samples showed agonistic activity for the ERα. Further investigations demonstrated anti-androgenic activity in the polar fraction of 9 out of 10 samples while no apolar extracts scored positive. The culprit chemicals causing the measured antagonistic activity in AR CALUX was investigated through liquid chromatography fractionation coupled to bioanalysis and non-target screening involving UHPLC-Q-TOF-MS/MS, using a pooled polar extract. The analysis revealed that the measured anti-androgenic biological activity could not be explained by the presence of endogenous hormones nor their metabolites. We have demonstrated that human milk of Norwegian mothers contained anti-androgenic activity which is most likely associated with the presence of anthropogenic polar EDCs without direct interferences from natural sex hormones. These findings warrant a larger scale investigation into endocrine biological activity in human milk, as well as exploring the chemical sources of the activity and their potential effects on health of the developing infant