Emulating the early phases of human tooth development in vitro

Functional in vitro models emulating the physiological processes of human organ formation are invaluable for future research and the development of regenerative therapies. Here, a developmentally inspired approach is pursued to reproduce fundamental steps of human tooth organogenesis in vitro using human dental pulp cells. Similar to the in vivo situation of tooth initiating mesenchymal condensation, a 3D self-organizing culture was pursued resulting in an organoid of the size of a human tooth germ with odontogenic marker expression. Furthermore, the model is capable of epithelial invagination into the condensed mesenchyme, mimicking the reciprocal tissue interactions of human tooth development. Comprehensive transcriptome analysis revealed activation of well-studied as well as rather less investigated signaling pathways implicated in human tooth organogenesis, such as the Notch signaling. Early condensation in vitro revealed a shift to the TGFß signal transduction pathway and a decreased RhoA small GTPase activity, connected to the remodeling of the cytoskeleton and actin-mediated mechanotransduction. Therefore, this in vitro model of tooth development provides a valuable model to study basic human developmental mechanisms.DFG, 414044773, Open Access Publizieren 2019 - 2020 / Technische Universität Berli

Per- and poly-fluoroalkyl substances (PFASs) in follicular fluid from women experiencing infertility in Australia

Per- and poly-fluoroalkyl substances (PFASs) have been widely used and detected in human matrices. Evidence that PFAS exposure may be associated with adverse human reproductive health effects exists, however, data is limited. The use of a human matrix such as follicular fluid to determine chemical exposure, along with reproductive data will be used to investigate if there is a relationship between PFAS exposure and human fertility. Objective: This study aims to: (1) assess if associations exist between PFAS concentrations and/or age and fertilisation rate (as determined in follicular fluid of women in Australia who received assisted reproductive treatment (ART)); and (2) assess if associations exist between PFAS concentrations and infertility aetiology. Methods: Follicular fluids were originally collected from participants who underwent fully stimulated ART treatment cycles at an in vitro fertilisation (IVF) clinic in the period 2006–2009 and 2010–11 in Queensland, Australia. The samples were available for analysis of 32 PFASs including perfluorooctane sulfonate (PFOS), perfluorooctanoic acid (PFOA), perfluorohexane sulfonate (PFHxS), and perfluorononanoic acid (PFNA) using high performance liquid chromatography tandem mass spectrometry (HPLC-MS/MS). 97 samples were matched with limited demographic data (age and fertilisation rate) and five infertility factors (three known female factors): 1) endometriosis, 2) polycystic ovarian syndrome (PCOS), and 3) genital tract infections - tubal/pelvic inflammation disease; as well as 4) male factor, and 5) idiopathic or unknown from either males or females. SPSS was used for linear regression analysis. Results: PFASs were detected in all follicular fluid samples with the mean concentrations of PFOS and PFOA, 4.9, and 2.4 ng/ml, respectively. A lower fertilisation rate was observed at higher age when age was added as a covariate, but there was no relationship between PFAS concentrations and fertilisation rate. There were few statistically significant associations between PFAS concentrations in follicular fluid and infertility factors. Log-transformed PFHxS concentrations were lower in females with endometriosis (factor 1) than in women who had reported ‘male factors’ as a reason of infertility, while PFHpA was higher in women who had infertile due to female factors (factor 1–3) compared to those who had infertile due to male factor. Conclusion: PFASs were detected in follicular fluid of Australian women who had been treated at an IVF clinic. PFAS exposure found in follicular fluids is linked to increased risk of some infertility factors, and increased age was associated with decreased fertilisation rate in our data. But there was no relationship between PFAS and ferlitisation rate. Further large-scale investigations of PFAS and health effects including infertility are warranted

Leaching and bioavailability of selected perfluoroalkyl acids (PFAAs) from soil contaminated by firefighting activities

Historical usage of aqueous film-forming foam (AFFF) at firefighting training grounds (FTGs) is a potential source of perfluoroalkyl acids (PFAAs) to the surrounding environment. In this study the leaching of PFAAs from field contaminated soil and their uptake into biota was investigated. Soil was sampled from FTGs at two airports and the total as well as the leachable concentration of 12 PFAAs was determined. A greenhouse study was carried out to investigate the uptake of PFAAs from soils into earthworms (Eisenia fetida) and wheat grass (Elymus scaber). Perfluorooctane sulfonate (PFOS) and perfluorohexane sulfonate (PFHxS) were the most dominant PFAAs in all soils samples, with concentrations of PFOS reaching 13,400 ng/g. Leachable concentrations of PFOS and PFHxS reached up to 550 μg/L and 22 μg/L, respectively. In earthworms concentrations of PFOS reached 65,100 ng/g after a 28-day exposure period, while in wheat grass the highest concentration was measured for uptake of PFHxS (2,800 ng/g) after a 10-week growth-period. Bioaccumulation factors (BAFs) for earthworms ranged from 0.1 for perfluorohexanoic acid (PFHxA) to 23 for perfluorododecanoic acid (PFDoA) and initially showed a decreasing trend with increasing perfluoroalkyl chain length, followed by an increase with increasing perfluoroalkyl chain length for perfluoroalkyl carboxylic acids (PFCAs). In wheat grass the highest BAF was found for perfluorobutanoic acid (BAF = 70), while the lowest was observed for perfluorononanoic acid (BAF = 0.06). BAFs in wheat grass decreased with increasing perfluoroalkyl chain length for both PFCAs and perfluoroalkyl sulfonic acids (PFSAs). The results show that PFAAs readily leach from impacted soils and are bioaccumulated into earthworms and plants in an analyte dependent way. This shows considerable potential for PFAAs to move away from the original source either by leaching or uptake into ecological receptors, which may be a potential entry route into the terrestrial foodweb

A novel contact assay for testing aryl hydrocarbon receptor (AhR)-mediated toxicity of chemicals and whole sediments in zebrafish (Danio rerio) embryos

The European Water Framework Directive aims to achieve a good ecological and chemical status in surface waters until 2015. Sediment toxicology plays a major role in this intention as sediments can act as a secondary source of pollution. In order to fulfill this legal obligation, there is an urgent need to develop whole-sediment exposure protocols, since sediment contact assays represent the most realistic scenario to simulate in situ exposure conditions. Therefore, in the present study, a vertebrate sediment contact assay to determine aryl hydrocarbon receptor (AhR)-mediated activity of particle-bound pollutants was developed. Furthermore, the activity and the expression of the CYP1 family in early life stages of zebrafish after exposure to freeze-dried sediment samples were investigated. In order to validate the developed protocol, effects of β-naphthoflavone and three selected sediment on zebrafish embryos were investigated. Results documented clearly AhR-mediated toxicity after exposure to β-naphthoflavone (β-NF) and to the sediment from the Vering canal. Upregulation of mRNA levels was observed for all investigated sediment samples. The highest levels of all investigated cyp genes (cyp1a, cyp1b1, cyp1c1, and cyp1c2) were recorded after exposure to the sediment sample of the Vering canal. In conclusion, the newly developed sediment contact assay can be recommended for the investigation of dioxin-like activities of single substances and the bioavailable fraction of complex environmental samples. Moreover, the exposure of whole zebrafish embryos to native (freeze-dried) sediment samples represents a highly realistic and ecologically relevant exposure scenario

A novel contact assay for testing aryl hydrocarbon receptor (AhR)-mediated toxicity of chemicals and whole sediments in zebrafish (Danio rerio) embryos

The European Water Framework Directive aims to achieve a good ecological and chemical status in surface waters until 2015. Sediment toxicology plays a major role in this intention as sediments can act as a secondary source of pollution. In order to fulfill this legal obligation, there is an urgent need to develop whole-sediment exposure protocols, since sediment contact assays represent the most realistic scenario to simulate in situ exposure conditions. Therefore, in the present study, a vertebrate sediment contact assay to determine aryl hydrocarbon receptor (AhR)-mediated activity of particle-bound pollutants was developed. Furthermore, the activity and the expression of the CYP1 family in early life stages of zebrafish after exposure to freeze-dried sediment samples were investigated. In order to validate the developed protocol, effects of β-naphthoflavone and three selected sediment on zebrafish embryos were investigated. Results documented clearly AhR-mediated toxicity after exposure to β-naphthoflavone (β-NF) and to the sediment from the Vering canal. Upregulation of mRNA levels was observed for all investigated sediment samples. The highest levels of all investigated cyp genes (cyp1a, cyp1b1, cyp1c1, and cyp1c2) were recorded after exposure to the sediment sample of the Vering canal. In conclusion, the newly developed sediment contact assay can be recommended for the investigation of dioxin-like activities of single substances and the bioavailable fraction of complex environmental samples. Moreover, the exposure of whole zebrafish embryos to native (freeze-dried) sediment samples represents a highly realistic and ecologically relevant exposure scenario