Effects of the mycotoxin patulin at the level of nuclear receptor transcriptional activity and steroidogenesis in vitro

Patulin (PAT) is a mycotoxin produced by various species of fungi, with Penicillium expansum being the most commonly occurring. Apples and apple products are the main sources of PAT contamination. This mycotoxin has been shown to induce toxic effects in animals, a few of which include reproductive toxicity and interference with the endocrine system. Here the endocrine disrupting potential of PAT has been investigated in vitro to identify disruption at the level of oestrogen, androgen, progestagen and glucocorticoid nuclear receptor transcriptional activity, and to assess interferences in estradiol, testosterone and progesterone steroid hormone production. At the receptor level, 0.5-5000ng/ml (0.0032-32μM) PAT did not appear to induce any specific (ant) agonistic responses in reporter gene assays (RGAs); however, nuclear transcriptional activity was affected. A >6 fold increase in the glucocorticoid receptor transcriptional activity was observed following treatment with 5000ng/ml PAT in the presence of cortisol. At the hormone production level, despite cytotoxicity being observed after treatment with 5000ng/ml PAT, estradiol levels had increased >2 fold. At 500ng/ml PAT treatment, an increase in progesterone and a decrease in testosterone production were observed. The findings of this study could be considered in assessing the health risks following exposure to PAT.

Validation of an ultra high performance liquid chromatography-tandem mass spectrometry method for detection and quantitation of 19 endocrine disruptors in milk

An endocrine disruptor (ED) is an exogenous compound that interferes with the body's endocrine system. Exposure to EDs may result in adverse health effects such as infertility and cancer. EDs are composed of a vast group of chemicals including compounds of natural origin such as phytoestrogens or mycotoxins and a wide range of man-made chemicals such as pesticides. Synthetic compounds may find their way into the food chain where a number of them can biomagnify. Additionally, processing activities and food contact materials may add further to the already existing pool of food contaminants. Thus, our diet is considered to be one of the main exposure routes to EDs. Some precautionary legislation has already been introduced to control production and/or application of some persistent organic pollutants with ED characteristics. However, newly emerging EDs with bioaccumulative properties have recently been reported to appear at lower tiers of the food chain but have not been monitored at the grander scale. Milk and dairy products are a major component of our diet, thus it is important to monitor them for EDs. However, most methods developed to date are devoted to one group of compounds at a time. The UHPLC-MS/MS method described here has been validated according to EC decision 2002/657/EC and allows simultaneous extraction, detection, quantitation and confirmation of 19 EDs in milk. The method calibration range is between 0.50 and 20.0 μg kg with coefficients of determination above 0.99 for all analytes. Precision varied from 4.7% to 23.4% in repeatability and reproducibility studies. Established CCα and CCβ values (0.11-0.67 μg kg) facilitate fast, reliable, quantitative and confirmatory analysis of sub μg kg levels of a range of EDs in milk.

Disruption of vitellogenesis and spermatogenesis by triclabendazole (TCBZ) in a TCBZ-resistant isolate of Fasciola hepatica following incubation in vitro with a P-glycoprotein inhibitor

A study has been carried out to investigate whether the action of triclabendazole (TCBZ) against Fasciola hepatica is altered by inhibition of P-glycoprotein (Pgp)-linked drug efflux pumps. The Sligo TCBZ-resistant fluke isolate was used for these experiments and the Pgp inhibitor selected was R(+)-verapamil [R(+)-VPL]. In the first experiment, flukes were initially incubated for 2 h in R(+)-VPL (100 μ m), then incubated in R(+)-VPL+triclabendazole sulphoxide (TCBZ.SO) (50 μg mL-1, or 133·1 μ m) until flukes ceased movement (at 9 h post-treatment). In a second experiment, flukes were incubated in TCBZ.SO alone and removed from the incubation medium following cessation of motility (after 15 h). In the third experiment, flukes were incubated for 24 h in R(+)-VPL on its own. Changes to the testis tubules and vitelline follicles following drug treatment and following Pgp inhibition were assessed by means of light microscope histology and transmission electron microscopy. Incubation of the Sligo isolate in either R(+)-VPL or TCBZ.SO on their own had a limited impact on the morphology of the two tissues. Greater disruption was observed when the drugs were combined, in terms of the block in development of the spermatogenic and vitelline cells and the apoptotic breakdown of the remaining cells. Sperm formation was severely affected and abnormal. Large spaces appeared in the vitelline follicles and synthesis of shell protein was disrupted. The results of this study support the concept of altered drug efflux in TCBZ-resistant flukes and indicate that drug transporters may play a role in the development of drug resistance.

Observational Constraints on the Catastrophic Disruption Rate of Small Main Belt Asteroids

We have calculated 90% confidence limits on the steady-state rate of catastrophic disruptions of main belt asteroids in terms of the absolute magnitude at which one catastrophic disruption occurs per year  as a function of the post-disruption increase in brightness (Δm) and subsequent brightness decay rate (τ  ). The confidence limits were calculated using the brightest unknown main belt asteroid (V=18.5) detected with the Pan-STARRS1 (Pan-STARRS1) telescope. We measured the Pan-STARRS1’s catastrophic disruption detection efficiency over a 453-day interval using the Pan-STARRS moving object processing system (MOPS) and a simple model for the catastrophic disruption event’s photometric behavior in a small aperture centered on the catastrophic disruption event. We then calculated the  contours in the ranges from  and  encompassing measured values from known cratering and disruption events and our model’s predictions. Our simplistic catastrophic disruption model suggests that  and  which would imply that H_0≳28—strongly inconsistent withH_{0,B2005=23.26±0.02} predicted by Bottke et al. (Bottke, W.F., Durda, D.D., Nesvorný, D., Jedicke, R., Morbidelli, A., Vokrouhlický, D., Levison, H.F. [2005]. Icarus, 179, 63–94.) using purely collisional models. However, if we assume that H_0=H0,B2005 our results constrain , inconsistent with our simplistic impact-generated catastrophic disruption model. We postulate that the solution to the discrepancy is that >99% of main belt catastrophic disruptions in the size range to which this study was sensitive (∼100 m) are not impact-generated, but are instead due to fainter rotational breakups, of which the recent discoveries of disrupted asteroids P/2013 P5 and P/2013 R3 are probable examples. We estimate that current and upcoming asteroid surveys may discover up to 10 catastrophic disruptions/year brighter than V=18.5.

Endocrine disruptor activity of multiple environmental food chain contaminants

Industrial chemicals, antimicrobials, drugs and personal care products have been reported as global pollutants which enter the food chain. Some of them have also been classified as endocrine disruptors based on results of various studies employing a number of in vitro/. vivo tests. The present study employed a mammalian reporter gene assay to assess the effects of known and emerging contaminants on estrogen nuclear receptor transactivation.Out of fifty-nine compounds assessed, estrogen receptor agonistic activity was observed for parabens (. n= 3), UV filters (. n= 6), phthalates (. n= 4) and a metabolite, pyrethroids (. n= 9) and their metabolites (. n= 3). Two compounds were estrogen receptor antagonists while some of the agonists enhanced 17β-estradiol mediated response.This study reports five new compounds (pyrethroids and their metabolites) possessing estrogen agonist activity and highlights for the first time that pyrethroid metabolites are of particular concern showing much greater estrogenic activity than their parent compounds.

An investigation of the endocrine disrupting potential of enniatin B using in vitro bioassays

Evidence that some of the fungal metabolites present in food and feed may act as potential endocrine disruptors is increasing. Enniatin B (ENN B) is among the emerging Fusarium mycotoxins known to contaminate cereals. In this study, the H295R and neonatal porcine Leydig cell (LC) models, and reporter gene assays (RGAs) have been used to investigate the endocrine disrupting activity of ENN B. Aspects of cell viability, cell cycle distribution, hormone production as well as the expression of key steroidogenic genes were assessed using the H295R cell model. Cell viability and hormone production levels were determined in the LC model, while cell viability and steroid hormone nuclear receptor transcriptional activity were measured using the RGAs. ENN B (0.01–100 μM) was cytotoxic in the H295R and LC models used; following 48 h incubation with 100 μM. Flow cytometry analysis showed that ENN B exposure (0.1–25 μM) led to an increased proportion of cells in the S phase at higher ENN B doses (>10 μM) while cells at G0/G1 phase were reduced. At the receptor level, ENN B (0.00156–15.6 μM) did not appear to induce any specific (ant) agonistic responses in reporter gene assays (RGAs), however cell viability was affected at 15.6 μM. Measurement of hormone levels in H295R cells revealed that the production of progesterone, testosterone and cortisol in exposed cells were reduced, but the level of estradiol was not significantly affected. There was a general reduction of estradiol and testosterone levels in exposed LC. Only the highest dose (100 μM) used had a significant effect, suggesting the observed inhibitory effect is more likely associated with the cytotoxic effect observed at this dose. Gene transcription analysis in H295R cells showed that twelve of the sixteen genes were significantly modulated (p < 0.05) by ENN B (10 μM) compared to the control. Genes HMGR, StAR, CYP11A, 3βHSD2 and CYP17 were downregulated, whereas the expression of CYP1A1, NR0B1, MC2R, CYP21, CYP11B1, CYP11B2 and CYP19 were upregulated. The reduction of hormones and modulation of genes at the lower dose (10 μM) in the H295R cells suggests that adrenal endocrine toxicity is an important potential hazard.

In utero exposure to cigarette chemicals induces sex-specific disruption of one-carbon metabolism and DNA methylation in the human fetal liver

Background: Maternal smoking is one of the most important modifiable risk factors for low birthweight, which is strongly associated with increased cardiometabolic disease risk in adulthood. Maternal smoking reduces the levels of the methyl donor vitamin B12 and is associated with altered DNA methylation at birth. Altered DNA methylation may be an important mechanism underlying increased disease susceptibility; however, the extent to which this can be induced in the developing fetus is unknown.

Methods: In this retrospective study, we measured concentrations of cobalt, vitamin B12, and mRNA transcripts encoding key enzymes in the 1-carbon cycle in 55 fetal human livers obtained from 11 to 21 weeks of gestation elective terminations and matched for gestation and maternal smoking. DNA methylation was measured at critical regions known to be susceptible to the in utero environment. Homocysteine concentrations were analyzed in plasma from 60 fetuses.

Results: In addition to identifying baseline sex differences, we found that maternal smoking was associated with sex-specific alterations of fetal liver vitamin B12, plasma homocysteine and expression of enzymes in the 1-carbon cycle in fetal liver. In the majority of the measured parameters which showed a sex difference, maternal smoking reduced the magnitude of that difference. Maternal smoking also altered DNA methylation at the imprinted gene IGF2 and the glucocorticoid receptor (GR/NR3C1).

Conclusions: Our unique data strengthen studies linking in utero exposures to altered DNA methylation by showing, for the first time, that such changes are present in fetal life and in a key metabolic target tissue, human fetal liver. Furthermore, these data propose a novel mechanism by which such changes are induced, namely through alterations in methyl donor availability and changes in 1-carbon metabolism.

The frequency of gastrointestinal endocrine tumours in a well-defined population--Northern Ireland 1970-1985

The reported incidence of gastrointestinal endocrine tumours is variable. In Northern Ireland circumstances allowing such an assessment are favourable with a central diagnostic laboratory and register established to collect data on tumours from a well-defined population of 1.5 million people. From 1970 to 1985, 368 cases were recorded of which 85 per cent were carcinoid tumours. The annual incidence of carcinoid tumours was 1.3 per 100,000 of the population and the majority occurred in the appendix (61 per cent). No patients presented with the carcinoid syndrome. The annual incidence for other tumours was 0.12 per 100,000 for insulinomas; islet cell tumours of unknown type 0.07; Zollinger-Ellison syndrome 0.05; and multiple endocrine neoplasia (MEN) 0.05. There were two cases of VIPoma, one glucagonoma, one neurotensinoma and one tumour producing ACTH. It is possible that some tumours are more uncommon than others because of difficulty in diagnosis.

Disruption of SF3B1 results in deregulated expression and splicing of key genes and pathways in myelodysplastic syndrome hematopoietic stem and progenitor cells

The splicing factor SF3B1 is the most commonly mutated gene in the myelodysplastic syndrome (MDS), particularly in patients with refractory anemia with ring sideroblasts (RARS). We investigated the functional effects of SF3B1 disruption in myeloid cell lines: SF3B1 knockdown resulted in growth inhibition, cell cycle arrest and impaired erythroid differentiation and deregulation of many genes and pathways, including cell cycle regulation and RNA processing. MDS is a disorder of the hematopoietic stem cell and we thus studied the transcriptome of CD34 + cells from MDS patients with SF3B1 mutations using RNA sequencing. Genes significantly differentially expressed at the transcript andor exon level in SF3B1 mutant compared with wild-type cases include genes that are involved in MDS pathogenesis (ASXL1 and CBL), iron homeostasis and mitochondrial metabolism (ALAS2, ABCB7 and SLC25A37) and RNA splicingprocessing (PRPF8 and HNRNPD). Many genes regulated by a DNA damage-induced BRCA1-BCLAF1-SF3B1 protein complex showed differential expressionsplicing in SF3B1 mutant cases. This is the first study to determine the target genes of SF3B1 mutation in MDS CD34 + cells. Our data indicate that SF3B1 has a critical role in MDS by affecting the expression and splicing of genes involved in specific cellular processespathways, many of which are relevant to the known RARS pathophysiology, suggesting a causal link.

Fasciola hepatica:disruption of spermatogenesis by the microfilament inhibitor cytochalasin B

The distribution of actin filaments in the spermatogenic cells of Fasciola hepatica was determined using a fluorescent derivative of phalloidin. Actin was localised primarily in the region of separation of a secondary spermatogonium from a primary spermatogonium, in the inner faces at the centre of four-cell clusters of tertiary spermatogonia and in the cytophore region of spermatocyte and spermatid rosettes. The effect of the microfilament inhibitor cytochalasin B (100 micrograms/ml) on the ultrastructure of the spermatogenic cells was determined in vitro by transmission electron microscopy using tissue-slice material. Cytochalasin B treatment led to the formation of bi- and multinucleate cells, whose frequency increased with progressively longer incubation periods. Few typical rosettes of spermatocyte and spermatid cells were evident from 6 h onwards, being replaced by syncytial masses of cells. Spermatozoon formation became abnormal in the longer treatment periods, the spermatozoa containing variable numbers of axonemes and an altered distribution of cortical microtubules. Multiple axonemes were observed in the cytoplasm of spermatid cells. The results are discussed in relation to the established role of actin in the cytokinesis phase of cell division and to the effects of cytochalasin B on other tissues and organ systems within the fluke.

The Ultraviolet-Bright, Slowly Declining Transient PS1-11af as a Partial Tidal Disruption Event

We present the Pan-STARRS1 discovery of the long-lived and blue transient PS1-11af, which was also detected by Galaxy Evolution Explorer with coordinated observations in the near-ultraviolet (NUV) band. PS1-11af is associated with the nucleus of an early type galaxy at redshift z = 0.4046 that exhibits no evidence for star formation or active galactic nucleus activity. Four epochs of spectroscopy reveal a pair of transient broad absorption features in the UV on otherwise featureless spectra. Despite the superficial similarity of these features to P-Cygni absorptions of supernovae (SNe), we conclude that PS1-11af is not consistent with the properties of known types of SNe. Blackbody fits to the spectral energy distribution are inconsistent with the cooling, expanding ejecta of a SN, and the velocities of the absorption features are too high to represent material in homologous expansion near a SN photosphere. However, the constant blue colors and slow evolution of the luminosity are similar to previous optically selected tidal disruption events (TDEs). The shape of the optical light curve is consistent with models for TDEs, but the minimum accreted mass necessary to power the observed luminosity is only 0.002 M, which points to a partial disruption model. A full disruption model predicts higher bolometric luminosities, which would require most of the radiation to be emitted in a separate component at high energies where we lack observations. In addition, the observed temperature is lower than that predicted by pure accretion disk models for TDEs and requires reprocessing to a constant, lower temperature. Three deep non-detections in the radio with the Very Large Array over the first two years after the event set strict limits on the production of any relativistic outflow comparable to Swift J1644+57, even if off-axis.

An ultraviolet-optical flare from the tidal disruption of a helium-rich stellar core

The flare of radiation from the tidal disruption and accretion of a star can be used as a marker for supermassive black holes that otherwise lie dormant and undetected in the centres of distant galaxies. Previous candidate flares have had declining light curves in good agreement with expectations, but with poor constraints on the time of disruption and the type of star disrupted, because the rising emission was not observed. Recently, two `relativistic' candidate tidal disruption events were discovered, each of whose extreme X-ray luminosity and synchrotron radio emission were interpreted as the onset of emission from a relativistic jet. Here we report a luminous ultraviolet-optical flare from the nuclear region of an inactive galaxy at a redshift of 0.1696. The observed continuum is cooler than expected for a simple accreting debris disk, but the well-sampled rise and decay of the light curve follow the predicted mass accretion rate and can be modelled to determine the time of disruption to an accuracy of two days. The black hole has a mass of about two million solar masses, modulo a factor dependent on the mass and radius of the star disrupted. On the basis of the spectroscopic signature of ionized helium from the unbound debris, we determine that the disrupted star was a helium-rich stellar core.

Calmodulin disruption impacts growth and motility in juvenile liver fluke

Background: Deficiencies in effective flukicide options and growing issues with drug resistance make current strategies for liver fluke control unsustainable, thereby promoting the need to identify and validate new control targets in Fasciola spp. parasites. Calmodulins (CaMs) are small calcium-sensing proteins with ubiquitous expression in all eukaryotic organisms and generally use fluctuations in intracellular calcium levels to modulate cell signalling events. CaMs are essential for fundamental processes including the phosphorylation of protein kinases, gene transcription, calcium transport and smooth muscle contraction. In the blood fluke Schistosoma mansoni, calmodulins have been implicated in egg hatching, miracidial transformation and larval development. Previously, CaMs have been identified amongst liver fluke excretory-secretory products and three CaM-like proteins have been characterised biochemically from adult Fasciola hepatica, although their functions remain unknown.

Methods: In this study, we set out to investigate the biological function and control target potential of F. hepatica CaMs (FhCaMs) using RNAi methodology alongside novel in vitro bioassays.

Results: Our results reveal that: (i) FhCaMs are widely expressed in parenchymal cells throughout the forebody region of juvenile fluke; (ii) significant transcriptional knockdown of FhCaM1-3 was inducible by exposure to either long (~200 nt) double stranded (ds) RNAs or 27 nt short interfering (si) RNAs, although siRNAs were less effective than long dsRNAs; (iii) transient long dsRNA exposure-induced RNA interference (RNAi) of FhCaMs triggered transcript knockdown that persisted for ≥ 21 days, and led to detectable suppression of FhCaM proteins; (iv) FhCaM RNAi significantly reduced the growth of juvenile flukes maintained in vitro; (v) FhCaM RNAi juveniles also displayed hyperactivity encompassing significantly increased migration; (vi) both the reduced growth and increased motility phenotypes were recapitulated in juvenile fluke using the CaM inhibitor trifluoperazine hydrochloride, supporting phenotype specificity.

Conclusions: These data indicate that the Ca(2+)-modulating functions of FhCaMs are important for juvenile fluke growth and movement and provide the first functional genomics-based example of a growth-defect resulting from gene silencing in liver fluke. Whilst the phenotypic impacts of FhCaM silencing on fluke behaviour do not strongly support their candidature as new flukicide targets, the growth impacts encourage further consideration, especially in light of the speed of juvenile fluke growth in vivo.