Effects of endocrine disruptors in zebrafish (Danio rerio) as revealed with the fish sexual development test

So-called “endocrine disrupting chemicals” (EDCs) are ubiquitous in the environment, especially in aquatic systems close to industrial and agricultural areas. Disturbances of the hormonal balance between estrogens and androgens are one central target of EDCs. Especially during sexual differentiation of organisms, the consequences of such a misbalance may easily lead to population-relevant effects. Thus research into EDCs has been massively intensified over the last decades. Since, at international levels, there is high interest to establish validated test systems for the evaluation of endocrine effects, the OECD has initiated a progran to develop test guidelines for the detection of EDCs. Within this scope, the present work forms part of a validation exercise for the so-called “fish sexual development test” (FSDT), an extension of the existing OECD test guideline 210, the fish early life-stage toxicity test as an OECD guideline. The main idea is to establish an in vivo test for EDCs, which is relatively straightforward, cheap and on the one hand more rapid than full life-cycle tests, but, on the other hand, more sensible than the currently discussed 21-day-screening test. In the present study, the two EDCs prochloraz and 4-tert-pentylphenol were selected as exemplary substances to be tested in the FSDT. Prochloraz is a fungicide that inhibits the enzyme aromatase and therefore the synthesis of steroids. 4-tert-pentylphenol belongs to the group of alkylphenols, which are known to interact with the estrogen receptor, and, therefore, are classified as pseudoestrogens. Zebrafish (Danio rerio) were exposed to these chemicals in flow-through systems for 60 days post-hatch, a period in which zebrafish finalize their sexual differentiation. Within this sensitive phase the sexual development can easily be disturbed by EDCs. After exposure, the zebrafish were measured and weighed, the gonads were examined by means of histological analysis, and vitellogenin concentrations were measured in heads and tails via ELISA. Both chemicals caused a shift in sex ratio (prochloraz: masculinization; 4-tert-pentylphenol: feminization), underdevelopment of the gonads, inhibition of growth and altered vitellogenin production. For the two substances examined, the sex ratio and the evaluation of maturity stages were both the most sensitive and the most relevant endpoints. Thus, even though reproductive and population-relevant effects could not be documented by the FSDTs, the test clearly had at least predictive power for what could have been seen in full life-cycle tests. Therefore, the present study corrobates the conclusion that the FSDT is an adequate test system for the evaluation of EDCs and that its further validation as an OECD test guideline is definitely required

Zebrafish (Danio rerio) as a model to study developmental effects of endocrine disruption: molecular mechanisms, as well as persistence and reversibility of effects

The present study aimed to investigate the reversibility of endocrine disruption in zebrafish (Danio rerio) at different effect levels. For this purpose, three different endocrine disrupting chemicals (EDCs) were chosen according to their different modes of action. 17!- Ethinylestradiol (EE2), a semi-synthetic estrogen is the most popular substance in oral contraceptives and is regularly measured in surface waters. 17ß-trenbolone is an anabolic steroid, which binds with high affinity to the androgen receptor. The substance is used in industrial animal farming and sports as muscle growth promoter. Prochloraz is a popular fungicide with multiple modes of action. Its main effect on the endocrine system is that it inhibits the enzyme aromatase, which is essential for the conversion of androgens into estrogens. The effects of those three EDCs were assessed by performance of exposure experiments with developing zebrafish. After exposure of 60 days (from fertilized egg to sexual differentiation), half of the fish were continuously exposed until 100 days post hatch and the other half was held in clean water. In addition, this project focuses on assessing the correlation between different levels of biological organization. For this purpose, five effect levels with different ecological relevance were investigated: (1) population level: sex ratio; (2) individual level: growth; (3) organ/cell level: histology of gonads; (4) protein level: vitellogenin (VTG) induction and (5) mRNA level: aromatase (cyp19b) expression in brain. The three different EDCs investigated in this study showed strong impact on the sexual development of zebrafish at all effect levels at environmentally relevant concentrations. For trenbolone and prochloraz we could only find tendencies of reversibility, most effects remained unchanged after 40 days of depuration in clean water. Despite different underlying mechanisms, these substances produce an irreversible and considerable drift of the sex ratio towards males, as well as permanent effects on growth, VTG and aromatase levels. A clear reversibility of those effects could only be shown for EE2. Even at population level the impact of the semi-synthetic estrogen was reversible. These results show that the sexual development of zebrafish is a fragile process that can easily be disrupted permanently by substances that are found in the environment. Moreover, the results indicate that even a periodic exposure to those EDCs can cause severe impairment for wildlife and humans

Adverse effects in the fish embryo acute toxicity (FET) test : a catalogue of unspecific morphological changes versus more specific effects in zebrafish (Danio rerio) embryos

Funding Open Access funding enabled and organized by Projekt DEAL. This project has received funding from the European Union’s Horizon 2020 research and innovation programme under Grand agreement No 681102.Publisher PD

Poly(ADP-RIBOSE) polymerase-1 (Parp-1) antagonizes topoisomerase I-dependent recombination stimulation by P53

PARP-1 interacts with and poly(ADP-ribosyl)ates p53 and topoisomerase I, which both participate in DNA recombination. Previously, we showed that PARP-1 downregulates homology-directed double-strand break (DSB) repair. We also discovered that, despite the well-established role of p53 as a global suppressor of error-prone recombination, p53 enhances homologous recombination (HR) at the RARα breakpoint cluster region (bcr) comprising topoisomerase I recognition sites. Using an SV40-based assay and isogenic cell lines differing in the p53 and PARP-1 status we demonstrate that PARP-1 counteracts HR enhancement by p53, although DNA replication was largely unaffected. When the same DNA element was integrated in an episomal recombination plasmid, both p53 and PARP-1 exerted anti-recombinogenic rather than stimulatory activities. Strikingly, with DNA substrates integrated into cellular chromosomes, enhancement of HR by p53 and antagonistic PARP-1 action was seen, very similar to the HR of viral minichromosomes. siRNA-mediated knockdown revealed the essential role of topoisomerase I in this regulatory mechanism. However, after I-SceI-meganuclease-mediated cleavage of the chromosomally integrated substrate, no topoisomerase I-dependent effects by p53 and PARP-1 were observed. Our data further indicate that PARP-1, probably through topoisomerase I interactions rather than poly(ADP-ribosyl)ation, prevents p53 from stimulating spontaneous HR on chromosomes via topoisomerase I activity

Integration of mobile health into sickle cell disease care to increase hydroxyurea utilization: Protocol for an efficacy and implementation study

BACKGROUND: Hydroxyurea prevents disease complications among patients with sickle cell disease (SCD). Although its efficacy has been endorsed by the National Health Lung and Blood Institute evidence-based guidelines, its adoption is low, both by patients with SCD and providers. Mobile health (mHealth) apps provide benefits in improving medication adherence and self-efficacy among patients with chronic diseases and have facilitated prescription among medical providers. However, mHealth has not been systematically tested as a tool to increase hydroxyurea adherence nor has the combination of mHealth been assessed at both patient and provider levels to increase hydroxyurea utilization. OBJECTIVE: This study aims to increase hydroxyurea utilization through a combined two-level mHealth intervention for both patients with SCD and their providers with the goals of increasing adherence to hydroxyurea among patients and improve hydroxyurea prescribing behavior among providers. METHODS: We will test the efficacy of 2 mHealth interventions to increase both patient and provider utilization and knowledge of hydroxyurea in 8 clinical sites of the NHLBI-funded Sickle Cell Disease Implementation Consortium (SCDIC). The patient mHealth intervention, InCharge Health, includes multiple components that address memory, motivation, and knowledge barriers to hydroxyurea use. The provider mHealth intervention, Hydroxyurea Toolbox (HU Toolbox), addresses the clinical knowledge barriers in prescribing and monitoring hydroxyurea. The primary hypothesis is that among adolescents and adults with SCD, adherence to hydroxyurea, as measured by the proportion of days covered (the ratio of the number of days the patient is covered by the medication to the number of days in the treatment period), will increase by at least 20% after 24 weeks of receiving the InCharge Health app, compared with their adherence at baseline. As secondary objectives, we will (1) examine the change in health-related quality of life, acute disease complications, perceived health literacy, and perceived self-efficacy in taking hydroxyurea among patients who use InCharge Health and (2) examine potential increases in the awareness of hydroxyurea benefits and risks, appropriate prescribing, and perceived self-efficacy to correctly administer hydroxyurea therapy among SCD providers between baseline and 9 months of using the HU Toolbox app. We will measure the reach, adoption, implementation, and maintenance of both the InCharge Health and the HU Toolbox apps using the reach, effectiveness, adoption, implementation, and maintenance framework and qualitatively evaluate the implementation of both mHealth interventions. RESULTS: The study is currently enrolling study participants. Recruitment is anticipated to be completed by mid-2021. CONCLUSIONS: If this two-level intervention, that is, the combined use of InCharge Health and HU Toolbox apps, demonstrates efficacy in increasing adherence to hydroxyurea and prescribing behavior in patients with SCD and their providers, respectively, both apps will be offered to other institutions outside the SCDIC through a future large-scale implementation-effectiveness study. TRIAL REGISTRATION: ClinicalTrials.gov NCT04080167; https://clinicaltrials.gov/ct2/show/NCT04080167. INTERNATIONAL REGISTERED REPORT IDENTIFIER (IRRID): DERR1-10.2196/16319

The sensitivity of the zebrafish embryo coiling assay for the detection of neurotoxicity by compounds with diverse modes of action

Open Access via the Springer Agreement Funding: This project has received funding from the European Union’s Horizon 2020 research and innovation programme under grant agreement No 681002 (EU-ToxRisk).Peer reviewedPublisher PD

NF-κB regulates DNA double-strand break repair in conjunction with BRCA1–CtIP complexes

NF-κB is involved in immune responses, inflammation, oncogenesis, cell proliferation and apoptosis. Even though NF-κB can be activated by DNA damage via Ataxia telangiectasia-mutated (ATM) signalling, little was known about an involvement in DNA repair. In this work, we dissected distinct DNA double-strand break (DSB) repair mechanisms revealing a stimulatory role of NF-κB in homologous recombination (HR). This effect was independent of chromatin context, cell cycle distribution or cross-talk with p53. It was not mediated by the transcriptional NF-κB targets Bcl2, BAX or Ku70, known for their dual roles in apoptosis and DSB repair. A contribution by Bcl-xL was abrogated when caspases were inhibited. Notably, HR induction by NF-κB required the targets ATM and BRCA2. Additionally, we provide evidence that NF-κB interacts with CtIP–BRCA1 complexes and promotes BRCA1 stabilization, and thereby contributes to HR induction. Immunofluorescence analysis revealed accelerated formation of replication protein A (RPA) and Rad51 foci upon NF-κB activation indicating HR stimulation through DSB resection by the interacting CtIP–BRCA1 complex and Rad51 filament formation. Taken together, these results define multiple NF-κB-dependent mechanisms regulating HR induction, and thereby providing a novel intriguing explanation for both NF-κB-mediated resistance to chemo- and radiotherapies as well as for the sensitization by pharmaceutical intervention of NF-κB activation

Impact of the COVID-19 pandemic on the implementation of mobile health to improve the uptake of hydroxyurea in patients with sickle cell disease: Mixed methods study

BACKGROUND: Hydroxyurea therapy is effective for reducing complications related to sickle cell disease (SCD) and is recommended by National Health Lung and Blood Institute care guidelines. However, hydroxyurea is underutilized, and adherence is suboptimal. We wanted to test a multilevel mobile health (mHealth) intervention to increase hydroxyurea adherence among patients and improve prescribing among providers in a multicenter clinical trial. In the first 2 study sites, participants were exposed to the early phases of the COVID-19 pandemic, which included disruption to their regular SCD care. OBJECTIVE: We aimed to describe the impact of the COVID-19 pandemic on the implementation of an mHealth behavioral intervention for improving hydroxyurea adherence among patients with SCD. METHODS: The first 2 sites initiated enrollment 3 months prior to the start of the pandemic (November 2019 to March 2020). During implementation, site A clinics shut down for 2 months and site B clinics shut down for 9 months. We used the reach, effectiveness, adoption, implementation, and maintenance (RE-AIM) framework to evaluate the implementation and effectiveness of the intervention. mHealth implementation was assessed based on patients\u27 daily app use. Adherence to hydroxyurea was calculated as the proportion of days covered (PDC) from prescription records over the first 12 and 24 weeks after implementation. A linear model examined the relationship between app usage and PDC change, adjusting for baseline PDC, lockdown duration, and site. We conducted semistructured interviews with patients, health care providers, administrators, and research staff to identify factors associated with mHealth implementation and effectiveness. We used a mixed methods approach to investigate the convergence of qualitative and quantitative findings. RESULTS: The percentage of patients accessing the app decreased after March 15, 2020 from 86% (n=55) to 70% (n=45). The overall mean PDC increase from baseline to week 12 was 4.5% (P=.32) and to week 24 was 1.5% (P=.70). The mean PDC change was greater at site A (12 weeks: 20.9%; P=.003; 24 weeks: 16.7%; P=.01) than site B (12 weeks: -8.2%; P=.14; 24 weeks: -10.3%; P=.02). After adjustment, PDC change was 13.8% greater in those with increased app use after March 15, 2020. Interview findings indicated that site B\u27s closure during COVID-19 had a greater impact, but almost all patients reported that the InCharge Health app helped support more consistent medication use. CONCLUSIONS: We found significant impacts of the early clinic lockdowns, which reduced implementation of the mHealth intervention and led to reduced patient adherence to hydroxyurea. However, disruptions were lower among participants who experienced shorter clinic lockdowns and were associated with higher hydroxyurea adherence. Investigation of added strategies to mitigate the effects of care interruptions during major emergencies (eg, patient coaching and health navigation) may insulate the implementation of interventions to increase medication adherence. TRIAL REGISTRATION: ClinicalTrials.gov NCT04080167; https://clinicaltrials.gov/ct2/show/NCT04080167. INTERNATIONAL REGISTERED REPORT IDENTIFIER (IRRID): RR2-10.2196/16319

Estimating the incidence of acute infectious intestinal disease in the community in the UK:A retrospective telephone survey

Objectives: To estimate the burden of intestinal infectious disease (IID) in the UK and determine whether disease burden estimations using a retrospective study design differ from those using a prospective study design. Design/Setting: A retrospective telephone survey undertaken in each of the four countries comprising the United Kingdom. Participants were randomly asked about illness either in the past 7 or 28 days. Participants: 14,813 individuals for all of whom we had a legible recording of their agreement to participate Outcomes: Self-reported IID, defined as loose stools or clinically significant vomiting lasting less than two weeks, in the absence of a known non-infectious cause. Results: The rate of self-reported IID varied substantially depending on whether asked for illness in the previous 7 or 28 days. After standardising for age and sex, and adjusting for the number of interviews completed each month and the relative size of each UK country, the estimated rate of IID in the 7-day recall group was 1,530 cases per 1,000 person-years (95% CI: 1135 – 2113), while in the 28-day recall group it was 533 cases per 1,000 person-years (95% CI: 377 – 778). There was no significant variation in rates between the four countries. Rates in this study were also higher than in a related prospective study undertaken at the same time. Conclusions: The estimated burden of disease from IID varied dramatically depending on study design. Retrospective studies of IID give higher estimates of disease burden than prospective studies. Of retrospective studies longer recall periods give lower estimated rates than studies with short recall periods. Caution needs to be exercised when comparing studies of self-reported IID as small changes in study design or case definition can markedly affect estimated rates