Appropriate 'housekeeping' genes for use in expression profiling the effects of environmental estrogens in fish

BACKGROUND: Attempts to develop a mechanistic understanding of the effects of environmental estrogens on fish are increasingly conducted at the level of gene expression. Appropriate application of real-time PCR in such studies requires the use of a stably expressed 'housekeeping' gene as an internal control to normalize for differences in the amount of starting template between samples. RESULTS: We sought to identify appropriate genes for use as internal controls in experimental treatments with estrogen by analyzing the expression of eight functionally distinct 'housekeeping' genes (18S ribosomal RNA [18S rRNA], ribosomal protein l8 [rpl8], elongation factor 1 alpha [ef1a], glucose-6-phosphate dehydrogenase [g6pd], beta actin [bactin], glyceraldehyde-3-phosphate dehydrogenase [gapdh], hypoxanthine phosphoribosyltransferase 1 [hprt1], and tata box binding protein [tbp]) following exposure to the environmental estrogen, 17α-ethinylestradiol (EE(2)), in the fathead minnow (Pimephales promelas). Exposure to 10 ng/L EE(2 )for 21 days down-regulated the expression of ef1a, g6pd, bactin and gapdh in the liver, and bactin and gapdh in the gonad. Some of these effects were gender-specific, with bactin in the liver and gapdh in the gonad down-regulated by EE(2 )in males only. Furthermore, when ef1a, g6pd, bactin or gapdh were used for normalization, the hepatic expression of two genes of interest, vitellogenin (vtg) and cytochrome P450 1A (cyp1a) following exposure to EE(2 )was overestimated. CONCLUSION: Based on the data presented, we recommend 18S rRNA, rpl8, hprt1 and/or tbp, but not ef1a, g6pd, bactin and/or gapdh, as likely appropriate internal controls in real-time PCR studies of estrogens effects in fish. Our studies show that pre-validation of control genes considering the scope and nature of the experiments to be performed, including both gender and tissue type, is critical for accurate assessments of the effects of environmental estrogens on gene expression in fish

Video Measurements and Analysis of Surface Gravity Waves in Shallow Water

This paper discusses a shallow-water wave height measurement method that uses high definition video cameras to image a water surface wave patch. Wave height time series are extracted from water surface video sequences. Wave features such as the wavelength distribution and energy contained in a wave patch (W/m2) were obtained by analyzing the extracted wave height time series and expressing the wind-driven wave energy as a wave energy spectrum. A Weibull probability distribution was used as the mathematical form of the energy spectrum. Wave spectra are used as input to a wave patch simulation model that generates simulated wind-driven wave images. The measurement protocol is inexpensive, easy to implement, and useful to calibrate and validate wind-driven wave models. The protocol is used to understand resuspension of bottom muds due to wind waves in shallow waters. Scaled staff gauges made of polyvinyl chloride (PVC) materials are placed in shallow water and imaged at 30 Hz followed by frame based image analysis to extract wave height time series. Wave spectra calculated using the fast Fourier transform (FFT) results in a Weibull probability distribution function (WPDF) energy spectrum. The estimated wave spectrum is used to estimate wave energy in W/m2 followed by generation of wave patch simulations of the water surface. Simulated wave patches are compared with the sensor-based wave patch video measurements. Sensitivity analysis of coefficients α and β in the model are used to adjust the synthetic wave images to measured wave patch images. The approach allows one to obtain an estimate of the energy (W/m2) transferred from the local wind field to a water surface gravity wave patch

Endocrine disruption in aquatic systems: up-scaling research to address ecological consequences

Endocrine-disrupting chemicals (EDCs) can alter biological function in organisms at environmentally relevant concentrations and are a significant threat to aquatic biodiversity, but there is little understanding of exposure consequences for populations, communities and ecosystems. The pervasive nature of EDCs within aquatic environments and their multiple sub-lethal effects make assessments of their impact especially important but also highly challenging. Herein, we review the data on EDC effects in aquatic systems focusing on studies assessing populations and ecosystems, and including how biotic and abiotic processes may affect, and be affected by, responses to EDCs. Recent research indicates a significant influence of behavioural responses (e.g. enhancing feeding rates), transgenerational effects and trophic cascades in the ecological consequences of EDC exposure. In addition, interactions between EDCs and other chemical, physical and biological factors generate uncertainty in our understanding of the ecological effects of EDCs within aquatic ecosystems. We illustrate how effect thresholds for EDCs generated from individual-based experimental bioassays of the types commonly applied using chemical test guidelines [e.g. Organisation for Economic Co-operation and Development (OECD)] may not necessarily reflect the hazards associated with endocrine disruption. We argue that improved risk assessment for EDCs in aquatic ecosystems urgently requires more ecologically oriented research as well as field-based assessments at population-, community- and food-web levels

Concentrating mixtures of neuroactive pharmaceuticals and altered neurotransmitter levels in the brain of fish exposed to a wastewater effluent

Fish can be exposed to a variety of neuroactive pharmaceuticals via the effluent discharges from wastewater treatment plants and concerns have arisen regarding their potential impacts on fish behaviour and ecology. In this study, we investigated the uptake of 14 neuroactive pharmaceuticals from a treated wastewater effluent into blood plasma and brain regions of roach (Rutilus rutilus) after exposure for 15 days. We show that a complex mixture of pharmaceuticals including, 6 selective serotonin reuptake inhibitors, a serotonin-noradrenaline reuptake inhibitor, 3 atypical antipsychotics, 2 tricyclic antidepressants and a benzodiazepine, concentrate in different regions of the brain including the telencephalon, hypothalamus, optic tectum and hindbrain of effluent-exposed fish. Pharmaceuticals, with the exception of nordiazepam, were between 3–40 fold higher in brain compared with blood plasma, showing these neuroactive drugs are readily uptaken, into brain tissues in fish. To assess for the potential for any adverse ecotoxicological effects, the effect ratio was calculated from human therapeutic plasma concentrations (HtPCs) and the measured or predicted fish plasma concentrations of pharmaceuticals. After accounting for a safety factor of 1000, the effect ratios indicated that fluoxetine, norfluoxetine, sertraline, and amitriptyline warrant prioritisation for risk assessment studies. Furthermore, although plasma concentrations of all the pharmaceuticals were between 33 and 5714-fold below HtPCs, alterations in serotonin, glutamate, acetylcholine and tryptophan concentrations were observed in different brain regions of effluent-exposed fish. This study highlights the importance of determining the potential health effects arising from the concentration of complex environmental mixtures in risk assessment studies

Assessing variation in the potential susceptibility of fish to pharmaceuticals, considering evolutionary differences in their physiology and ecology

Fish represent the planet's most diverse group of vertebrates and they can be exposed to a wide range of pharmaceuticals. For practical reasons, extrapolation of pharmaceutical effects from 'model' species to other fish species is adopted in risk assessment. Here, we critically assess this approach. First, we show that between 65% and 86% of human drug targets are evolutionarily conserved in 12 diverse fish species. Focusing on nuclear steroid hormone receptors, we further show that the sequence of the ligand binding domain that plays a key role in drug potency is highly conserved, but there is variation between species. This variation for the oestrogen receptor, however, does not obviously account for observed differences in receptor activation. Taking the synthetic oestrogen ethinyloestradiol as a test case, and using life-table-response experiments, we demonstrate significant reductions in population growth in fathead minnow and medaka, but not zebrafish, for environmentally relevant exposures. This finding contrasts with zebrafish being ranked as more ecologically susceptible, according to two independent life-history analyses. We conclude that while most drug targets are conserved in fish, evolutionary divergence in drug-target activation, physiology, behaviour and ecological life history make it difficult to predict population-level effects. This justifies the conventional use of at least a 10× assessment factor in pharmaceutical risk assessment, to account for differences in species susceptibility.Swedish Foundation for Strategic Environmental Research (Mistra)UK Natural Environment Research Council (NERC)AstraZeneca's Global SHE Research Programm

Assessing the Sensitivity of Different Life Stages for Sexual Disruption in Roach (Rutilus rutilus) Exposed to Effluents from Wastewater Treatment Works

Surveys of U.K. rivers have shown a high incidence of sexual disruption in populations of wild roach (Rutilus rutilus) living downstream from wastewater treatment works (WwTW), and the degree of intersex (gonads containing both male and female structural characteristics) has been correlated with the concentration of effluent in those rivers. In this study, we investigated feminized responses to two estrogenic WwTWs in roach exposed for periods during life stages of germ cell division (early life and the postspawning period). Roach were exposed as embryos from fertilization up to 300 days posthatch (dph; to include the period of gonadal sex differentiation) or as postspawning adult males, and including fish that had received previous estrogen exposure, for either 60 or 120 days when the annual event of germ cell proliferation occurs. Both effluents induced vitellogenin synthesis in both life stages studied, and the magnitude of the vitellogenic responses paralleled the effluent content of steroid estrogens. Feminization of the reproductive ducts occurred in male fish in a concentration-dependent manner when the exposure occurred during early life, but we found no effects on the reproductive ducts in adult males. Depuration studies (maintenance of fish in clean water after exposure to WwTW effluent) confirmed that the feminization of the reproductive duct was permanent. We found no evidence of ovotestis development in fish that had no previous estrogen exposure for any of the treatments. In wild adult roach that had previously received exposure to estrogen and were intersex, the degree of intersex increased during the study period, but this was not related to the immediate effluent exposure, suggesting a previously determined programming of ovotestis formation

Dosimetric comparison study between intensity modulated radiation therapy and three-dimensional conformal proton therapy for pelvic bone marrow sparing in the treatment of cervical cancer.

The objective was to compare intensity-modulated radiation therapy (IMRT) with 3D conformal proton therapy (3DCPT) in the treatment of cervical cancer. In particular, each technique's ability to spare pelvic bone marrow (PBM) was of primary interest in this study. A total of six cervical cancer patients (3 postoperative and 3 intact) were planned and analyzed. All plans had uniform 1.0 cm CTV-PTV margin and satisfied the 95% PTV with 100% isodose (prescription dose = 45 Gy) coverage. Dose-volume histograms (DVH) were analyzed for comparison. The overall PTV and PBM volumes were 1035.9 ± 192.2 cc and 1151.4 ± 198.3 cc, respectively. In terms of PTV dose conformity index (DCI) and dose homogeneity index (DHI), 3DCPT was slightly superior to IMRT with 1.00 ± 0.001, 1.01 ± 0.02, and 1.10 ± 0.02, 1.13 ± 0.01, respectively. In addition, 3DCPT demonstrated superiority in reducing lower doses (i.e., V30 or less) to PBM, small bowel and bladder. Particularly in PBM, average V10 and V20 reductions of 10.8% and 7.4% (p = 0.001 and 0.04), respectively, were observed. However, in the higher dose range, IMRT provided better sparing (> V30). For example, in small bowel and PBM, average reductions in V45 of 4.9% and 10.0% (p = 0.048 and 0.008), respectively, were observed. Due to its physical characteristics such as low entrance dose, spread-out Bragg peak and finite particle range of protons, 3DCPT illustrated superior target coverage uniformity and sparing of the lower doses in PBM and other organs. Further studies are, however, needed to fully exploit the benefits of protons for general use in cervical cancer

Microplastic ingestion by riverine macroinvertebrates

Although microplastics are a recognised pollutant in marine environments, less attention has been directed towards freshwater ecosystems despite their greater proximity to possible plastic sources. Here, we quantify the presence of microplastic particles (MPs) in river organisms upstream and downstream of five UK Wastewater Treatment Works (WwTWs). MPs were identified in approximately 50% of macroinvertebrate samples collected (Baetidae, Heptageniidae and Hydropsychidae) at concentrations up to 0.14 MP mg tissue−1 and they occurred at all sites. MP abundance was associated with macroinvertebrate biomass and taxonomic family, but MPs occurred independently of feeding guild and biological traits such as habitat affinity and ecological niche. There was no increase in plastic ingestion downstream of WwTW discharges averaged across sites, but MP abundance in macroinvertebrates marginally increased where effluent discharges contributed more to total runoff and declined with increasing river discharge. The ubiquity of microplastics within macroinvertebrates in this case study reveals a potential risk from MPs entering riverine food webs through at least two pathways, involving detritivory and filter-feeding, and we recommend closer attention to freshwater ecosystems in future research

Biological traits and the transfer of persistent organic pollutants through river food webs

Freshwater organisms remain at risk from bioaccumulation and biomagnification of persistent organic pollutants (POPs), but factors affecting their transfer through food webs are poorly understood. Here, we investigate transfer pathways of polychlorinated biphenyls, polybrominated diphenyl ethers, and organochlorine through a river food web, assessing the distribution and flux between basal resources (n = 3), macroinvertebrates (n = 22), and fish (n = 1). We investigate the effects of biological traits on the observed patterns and use trait-based models to predict POP bioaccumulation. Transfer pathways differed among POPs and traits such as habitat affinity, feeding behavior, and body size explained some variation in POP burdens between organisms. Trait-based models indicated that relationships between POPs, trophic transfers, and traits were relatively well conserved across a wider array of river food webs. Although providing more consistent predictions of POP bioaccumulation than steady-state models, variability in bioaccumulation across food webs limited the accuracy of trait-model predictions. As some of the first data to illustrate how ecological processes alter the flux of pollutants through river food webs, these results reveal important links between POPs and contrasting energetic pathways. These data also show the utility of trait-based methods in the assessment of persistent contaminants, but further field validations are required