Branching morphogenesis of the mouse mammary gland after exposure to benzophenone-3.

Pubertal mammary branching morphogenesis is a hormone-regulated process susceptible to exposure to chemicals with endocrine disruptive capacity, such as the UV-filter benzophenone-3 (BP3). Our aim was to assess whether intrauterine or in vitro exposure to BP3 modified the branching morphogenesis of the female mouse mammary gland. For this, pregnant mice were dermally exposed to BP3 (0.15 or 50 mg/kg/day) from gestation day (GD) 8.5 to GD18.5. Sesame oil treatment served as control. Changes of the mammary glands of the offspring were studied on postnatal day 45. Further, mammary organoids from untreated mice were cultured under branching induction conditions and exposed for 9 days to BP3 (1 × 10-6 M, 1 × 10-9 M, or 1 × 10-12 M with 0.01% ethanol as control) to evaluate the branching progression. Mice that were exposed to BP3 in utero showed decreased mRNA levels of progesterone receptor (PR) and WNT4. However, estradiol and progesterone serum levels, mammary histomorphology, proliferation, and protein expression of estrogen receptor alpha (ESR1) and PR were not significantly altered. Interestingly, direct exposure to BP3 in vitro also decreased the mRNA levels of PR, RANKL, and amphiregulin without affecting the branching progression. Most effects were found after exposure to 50 mg/kg/day or 1 × 10-6 M of BP3, both related to sunscreen application in humans. In conclusion, exposure to BP3 does not impair mammary branching morphogenesis in our models. However, BP3 affects PR transcriptional expression and its downstream mediators, suggesting that exposure to BP3 might affect other developmental stages of the mammary gland.

Single and mixture effects of bisphenol A and benzophenone-3 on in vitro T helper cell differentiation.

Immune homeostasis is key to guarantee that the immune system can elicit effector functions against pathogens and at the same time raise tolerance towards other antigens. A disturbance of this delicate balance may underlie or at least trigger pathologies. Endocrine disrupting chemicals (EDCs) are increasingly recognized as risk factors for immune dysregulation. However, the immunotoxic potential of specific EDCs and their mixtures is still poorly understood. Thus, we aimed to investigate the effect of bisphenol A (BPA) and benzophenone-3 (BP-3), alone and in combination, on in vitro differentiation of T helper (TH)17 cells and regulatory T (Treg) cells. Naïve T cells were isolated from mouse lymphoid tissues and differentiated into the respective TH population in the presence of 0.001-10 µM BP-3 and/or 0.01-100 µM BPA. Cell viability, proliferation and the expression of TH lineage specific transcription factors and cytokines was measured by flow cytometry and CBA/ELISA. Moreover, the transcription of hormone receptors as direct targets of EDCs was quantified by RT-PCR. We found that the highest BPA concentration adversely affected TH cell viability and proliferation. Moreover, the general differentiation potential of both TH populations was not altered in the presence of both EDCs. However, EDC exposure modulated the emergence of TH17 and Treg cell intermediate states. While BPA and BP-3 promoted the development of TH1-like TH17 cells under TH17-differentiating conditions, TH2-like Treg cells occurred under Treg polarization. Interestingly, differential effects could be observed in mixtures of the two tested compounds compared with the individual compounds. Notably, estrogen receptor ß expression was decreased under TH17-differentiating conditions in the presence of BPA and BP-3 as mixture. In conclusion, our study provides solid evidence for both, the immune disruptive potential and the existence of cumulative effects of real nature EDC mixtures on T cell in vitro differentiation.

A comprehensive battery of flow cytometric immunoassays for the in vitro testing of chemical effects in human blood cells.

Background: There is a growing need for immunological assays to test toxic and modulatory effects of chemicals. The assays should be easy to use, reproducible and superior to cell line-based assays. We have therefore developed a comprehensive portfolio of assays based on primary human blood cells that are suitable for testing chemical effects. Methods: The flow cytometry-based assays were designed to target a wide range of human peripheral blood mononuclear cells and whole blood, including T cells, NK cells, B cells, basophils and innate-like T cells such as γδT, MAIT and NKT cells. We have selected a set of activation markers for each immune cell, e.g: CD154 (T cells), CD137, CD107a (NK cells), CD63 (basophils), CD69, CD83 (B cells), CD69, IFN-γ (MAIT cells) and we selected cell specific stimuli: aCD3 antibodies (T cells); E. coli and cytokines IL-12/15/18 (MAIT cells); CpG ODN2006, R848 or aCD40 antibodies (B cells), fMLP or aFcϵR1 (basophils) or K562 cells (NK cells). Results: By selecting immune cell-specific markers and cell-specific stimuli, we were able to induce particular immune responses from the targeted immune cells. For example, the response to stimulation with anti-CD3 antibodies was in 36.8% of CD107a+CD8+ cells. Cytokine stimulation induced the production of IFN-γ in 30% of MAIT cells. After stimulation with E. coli, around 50% of MAIT cells produced TNF. About 40% of basophils responded to aFcƐR1 stimulation. Similar activation ranges were achieved in K562-stimulated NK cells. Conclusion: Our test portfolio covers the most relevant immune cells present in human blood, providing a solid basis for in vitro toxicity and immunomodulatory testing of chemicals. By using human blood, the natural composition of cells found in the blood can be determined and the effects of chemicals can be detected at the cellular level.