Effect-Based Analysis of Endocrine Disrupting Chemical Mixtures in Breast Milk and Possible Health Consequences for Human Infants.

The research described in this thesis investigated the amount and potential effects of EDCs present in human milk using samples derived from the Norwegian HUMIS cohort. This work focused on three main actors of the endocrine system, the estrogens, androgens, and thyroid hormones. The first aim of the thesis was to develop a suitable method to properly extract EDCs from human milk samples, focusing on both polar and apolar endocrine active contaminants. The second aim was to perform a pilot study on a limited number of breast milk samples, using the existing ERα and AR CALUX bioassays, in agonistic and antagonistic mode. This phase was also used to evaluate the nature and origin of the observed EDC activity, as well as the contribution of endogenous hormones to the measurements. The third goal was to measure the EDC activity in a larger set of human milk samples to evaluate the potential association between anti-androgenic EDCs and an androgen-dependent deformity: cryptorchidism. The last objective was the development and application of novel thyroid hormone-based bioassays, TRβ CALUX and TTR-TRβ CALUX assays. These bioassays were used to evaluate the impact of breast milk contaminants, including well-known PFAS (perfluorooctanoic acid or PFOA; perfluorooctane sulfonic acid or PFOS), on the thyroid system. The thesis is divided in two distinct parts: Chapter Two and Chapter Three focusing on sex steroid disruption, and Chapter Four and Chapter Five covering thyroid system interferences, respectively. In Chapter Two, the method developed to extract EDCs from breast milk samples, used throughout the thesis, is described. In this chapter, estrogenic, anti-estrogenic, androgenic, and anti-androgenic activities derived from ten human milk extracts were analyzed on the (anti-)ERα and (anti-)AR CALUX bioassays. To rule out the impact of endogenous hormones in the measured activity, a pooled breast milk sample presenting anti-androgenic activity was fractionated and screened for natural hormones and metabolites by means of non-target screening using time-of-flight mass spectrometry UHPLC-Q-TOF-MS/MS. Chapter Three describes a larger-scale study involving 199 participants and presents results of anti-androgenic EDC activity in breast milk samples. In this chapter, we investigated the potential association between anti-androgenic activity in mothers’ milk and the occurrence of cryptorchidism, an androgen-dependent deformity, in the offspring. Moreover, we estimated the overall extent of exposure of a nursing child to anti-androgenic EDCs via breastfeeding. In Chapter Four, the TRβ CALUX bioassay, allowing screening of TRβ disrupting activity, and the TTR-TRβ CALUX assay, designed to detect competing properties towards T4 for TTR binding, were evaluated using well-known reference compounds. The performance of the TTR-TRβ CALUX assay was also evaluated during a short pilot study involving water samples. In Chapter Five the newly validated TRβ and TTR-TRβ CALUX bioassays were further used to assess a set of thirteen PFAS, known to affect the TH system (Chang et al. 2008; Thibodeaux et al. 2003) for thyroid-disrupting activities. Subsequently, ten breast milk extracts, with known PFOS and PFOA concentrations, were analyzed on the same assays. In this chapter, the impact of PFOS and PFOA levels was weighed in respect to thyroid-disrupting activity in breast milk. Furthermore, the average exposure to thyroid-disrupting EDCs during the first year of life of a nursing infant through breastfeeding was estimated. Finally, Chapter Six reviews and discusses the most important outcomes of the studies regarding hormonal-disruption derived from the presence of EDCs in Norwegian breast milk samples. General conclusions and an outlook on future perspective are presented

Anti-androgenic compounds in breast milk and cryptorchidism among Norwegian boys in the HUMIS birth cohort

The prevalence of cryptorchidism has increased over the past decades, yet its origins remain poorly understood. Testis descent is dependent on androgens and likely affected by endocrine disrupting compounds (EDCs), targeting the androgen receptor (AR). We investigated the association between anti-androgenic activity, not derived from natural hormones, in maternal breast milk and impaired testis descent among boys. We performed a case-control study based on 199 breast milk samples from 94 mothers of cryptorchid boys and 105 random non-cryptorchid boys participating in the Norwegian HUMIS (Human Milk Study) cohort. For each participant, apolar, and polar fractions were extracted, and combined to reconstitute a mixture. Anti-androgenic activity was measured in all three fractions using the human cell-based in vitro anti-AR CALUX® assay and expressed in μg of flutamide equivalent, a well-known antiandrogen. Results from fraction analyses were compared among boys with cryptorchidism and controls using multiple logistic regression, controlling for appropriate confounders identified using a directed acyclic graph. Children's daily exposure to anti-androgenic EDCs through breastfeeding was estimated to 78 μg flutamide eq./kg of body weigh/day. The activity was higher in the polar fraction (1.48 ± 1.37 μg flutamide eq./g of milk) mainly representing non-persistent chemicals, in contrast to other fractions. However, the activity in the polar extracts was decreased when in mixtures with the apolar fraction, indicating synergistic interactions. No significant difference in the activity was observed according to cryptorchid status for polar, apolar or mixed breast milk fractions. The study showed anti-androgenic activity in nearly all human milk samples, and at levels higher than the advisory threshold. However, no significant association was observed between cryptorchidism and antiandrogenic activity measured in either polar, apolar, or mixture fractions derived from breast milk

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

Evaluation of a panel of in vitro methods for assessing thyroid receptor β and transthyretin transporter disrupting activities

We developed a thyroid testing panel to assess endocrine disrupting chemicals (EDCs) capacities to bind either the thyroid receptor β (TRβ) or the thyroid hormones transporter transthyretin (TTR). We first stably transfected a human U2OS cell line with TRβ and a luciferase reporter construct to develop the TRβ CALUX® reporter gene assay to assess chemicals’ potential to interact with TRβ. Secondly, we combined a TTR-binding assay with the TRβ CALUX (TTR-TRβ CALUX) and optimized the system to evaluate the competitive properties of EDCs towards T4 for TTR binding. Both systems were evaluated with a range of known thyroid-disrupting compounds. The agonistic/antagonistic TRβ CALUX successfully predicted 9/9 and 9/12 test compounds, respectively. The TTR-TRβ CALUX predicted 9/9 compounds and demonstrated competitive activities when analyzing waste water samples. We concluded that the proposed test battery is a promising screening method able to efficiently generate data on thyroid hormone interferences by chemicals

Autosomal recessive POLR1D mutation with decrease of TCOF1 mRNA is responsible for Treacher Collins syndrome

Purpose: Treacher Collins syndrome is a mandibulofacial dysostosis caused by mutations in genes involved in ribosome biogenesis and synthesis. TCOF1 mutations are observed in similar to 80% of the patients and are inherited in an autosomal dominant manner. Recently, two other genes have been reported in G, p.Leu55Val). This mutation is localized in a region encoding the dimerization domain of the RNA polymerase. It is supposed that this mutation impairs RNA polymerase, resulting in a lower amount of mature dimeric ribosomes. A functional analysis of the transcripts of TCOF1 by real-time quantitative reverse transcription-polymerase chain reaction was performed in the first family, demonstrating a 50% reduction in the index case, compatible with this hypothesis. Conclusion: This is the first report of POLR1D mutation being responsible for an autosomal recessive inherited Treacher Collins syndrome. These results reinforce the concept of genetic heterogeneity of Treacher Collins syndrome and underline the importance of combining clinical expertise and familial molecular analyses for appropriate genetic counseling

Treacher Collins syndrome: a clinical and molecular study based on a large series of patients.

International audiencePurpose - Treacher Collins/Franceschetti syndrome (TCS; OMIM 154500) is a disorder of craniofacial development belonging to the heterogeneous group of mandibulofacial dysostoses. TCS is classically characterized by bilateral mandibular and malar hypoplasia, downward-slanting palpebral fissures, and microtia. To date, three genes have been identified in TCS:,TCOF1, POLR1D, and POLR1C. Methods - We report a clinical and extensive molecular study, including TCOF1, POLR1D, POLR1C, and EFTUD2 genes, in a series of 146 patients with TCS. Phenotype-genotype correlations were investigated for 19 clinical features, between TCOF1 and POLR1D, and the type of mutation or its localization in the TCOF1 gene. Results - We identified 92/146 patients (63%) with a molecular anomaly within TCOF1, 9/146 (6%) within POLR1D, and none within POLR1C. Among the atypical negative patients (with intellectual disability and/or microcephaly), we identified four patients carrying a mutation in EFTUD2 and two patients with 5q32 deletion encompassing TCOF1 and CAMK2A in particular. Congenital cardiac defects occurred more frequently among patients with TCOF1 mutation (7/92, 8%) than reported in the literature. Conclusion - Even though TCOF1 and POLR1D were associated with extreme clinical variability, we found no phenotype-genotype correlation. In cases with a typical phenotype of TCS, 6/146 (4%) remained with an unidentified molecular defect