Characterisation and expression of β1-, β2- and β3-adrenergic receptors in the fathead minnow (Pimephales promelas)

This is the author’s version of a work that was accepted for publication in General and Comparative Endocrinology. Changes resulting from the publishing process, such as peer review, editing, corrections, structural formatting, and other quality control mechanisms may not be reflected in this document. Changes may have been made to this work since it was submitted for publication. A definitive version was subsequently published and may be accessed at the link below. Copyright © 2011 Elsevier B.V. All rights reserved.Complimentary DNAs for three beta-adrenergic receptors (βARs) were isolated and characterised in the fathead minnow. The encoded proteins of 402 (β(1)AR), 397 (β(2)AR) and 434 (β(3)AR) amino acids were homologous to other vertebrate βARs, and displayed the characteristic seven transmembrane helices of G Protein-coupled receptors. Motifs and amino acids shown to be important for ligand binding were conserved in the fathead minnow receptors. Quantitative RT-PCR revealed the expression of all receptors to be highest in the heart and lowest in the ovary. However, the β(1)AR was the predominant subtype in the heart (70%), and β(3)AR the predominant subtype in the ovary (53%). In the brain, β(1)AR expression was about 200-fold higher than that of β(2)- and β(3)AR, whereas in the liver, β(2)AR expression was about 20-fold and 100-fold higher than β(3)- and β(1)AR expression, respectively. Receptor gene expression was modulated by exposure to propranolol (0.001-1mg/L) for 21days, but not in a consistent, concentration-related manner. These results show that the fathead minnow has a beta-adrenergic receptor repertoire similar to that of mammals, with the molecular signatures required for ligand binding. An exogenous ligand, the beta-blocker propranolol, is able to alter the expression profile of these receptors, although the functional relevance of such changes remains to be determined. Characterisation of the molecular targets for beta-blockers in fish will aid informed environmental risk assessments of these drugs, which are known to be present in the aquatic environment.European Union as part of the ERAPharm project, Contract No. 511135 and NER

Developmental toxicity of metaldehyde in the embryos of Lymnaea stagnalis (Gastropoda: Pulmonata) co-exposed to the synergist piperonyl butoxide.

Metaldehyde is a widely used molluscicide in countries where damage to crops from slugs and snails is a major problem associated with warm and wet winters. In the UK it is estimated that over 8% of the area covered by arable crops is treated with formulated granular bait pellets containing metaldehyde as the main active ingredient. Metaldehyde is hydrophilic (log Kow=0.12), water soluble (200 mg·L(-1) at 17 °C) and has been detected in UK surface waters in the concentration range of typically 0.2-0.6 μg·L(-1) (maximum 2.7 μg·L(-1)) during 2008-2011. In the absence of chronic data on potential hazards to non-target freshwater molluscs, a laboratory study was conducted to investigate the impact of metaldehyde on embryo development in the gastropod Lymnaea stagnalis (RENILYS strain) and using zinc as a positive control. L. stagnalis embryos were exposed to metaldehyde under semi-static conditions at 20±1 °C and hatching success and growth (measured as shell height and intraocular distance) examined after 21 d. Exposure concentrations were verified using HPLC and gave 21 d (hatching)NOEC and (hatching)LOEC mean measured values of 36 and 116 mg MET·L(-1), respectively (equal to the 21 d (shell height)NOEC and (shell height)LOEC values). For basic research purposes, a second group of L. stagnalis embryos was co-exposed to metaldehyde and the pesticide synergist piperonyl butoxide (PBO). Co-exposure to the PBO (measured concentrations between 0.47-0.56 mg·L(-1)) reduced hatching success from 100% to 47% and resulted in a 30% reduction in embryo growth (shell height) in snail embryos co-exposed to metaldehyde at 34-36 mg·L(-1) over 21 d. In conclusion, these data suggest mollusc embryos may have some metabolic detoxication capacity for metaldehyde and further work is warranted to explore this aspect in order to support the recent initiative to include molluscs in the OECD test guideline programme

Promoting the 3Rs to enhance the OECD fish toxicity testing framework.

Fish toxicity testing has been conducted since the 1860's in order to help define safe levels of chemical contaminants in lakes, rivers and coastal waters. The historical emphasis on acute lethality testing of chemicals has more recently focussed on long term sublethal effects of chemicals on fish and their prey species. Fish toxicity testing is now embedded in much environment legislation on chemical safety while it is recognized that animal use should be Replaced, Reduced and Refined (the 3Rs) where possible. The OECD Fish Toxicity Testing Framework provides a useful structure with which to address the needs of environmental safety assessment whilst implementing the 3Rs. This commentary aims to promote the implementation of the recommendations of the OECD Fish Toxicity Testing Framework

Comparative metabolism as a key driver of wildlife species sensitivity to human and veterinary pharmaceuticals

Human and veterinary drug development addresses absorption, distribution, metabolism, elimination and toxicology (ADMET) of the Active Pharmaceutical Ingredient (API) in the target species. Metabolism is an important factor in controlling circulating plasma and target tissue API concentrations and in generating metabolites which are more easily eliminated in bile, faeces and urine. The essential purpose of xenobiotic metabolism is to convert lipid-soluble, non-polar and non-excretable chemicals into water soluble, polar molecules that are readily excreted. Xenobiotic metabolism is classified into Phase I enzymatic reactions (which add or expose reactive functional groups on xenobiotic molecules), Phase II reactions (resulting in xenobiotic conjugation with large water-soluble, polar molecules) and Phase III cellular efflux transport processes. The human-fish plasma model provides a useful approach to understanding the pharmacokinetics of APIs (e.g. diclofenac, ibuprofen and propranolol) in freshwater fish, where gill and liver metabolism of APIs have been shown to be of importance. By contrast, wildlife species with low metabolic competency may exhibit zero-order metabolic (pharmacokinetic) profiles and thus high API toxicity, as in the case of diclofenac and the dramatic decline of vulture populations across the Indian subcontinent. A similar threat looms for African Cape Griffon vultures exposed to ketoprofen and meloxicam, recent studies indicating toxicity relates to zero-order metabolism (suggesting P450 Phase I enzyme system or Phase II glucuronidation deficiencies). While all aspects of ADMET are important in toxicity evaluations, these observations demonstrate the importance of methods for predicting API comparative metabolism as a central part of environmental risk assessment

Pyrosequencing of Mytilus galloprovincialis cDNAs: tissue-specific expression patterns.

BACKGROUND: Mytilus species are important in marine ecology and in environmental quality assessment, yet their molecular biology is poorly understood. Molecular aspects of their reproduction, hybridisation between species, mitochondrial inheritance, skewed sex ratios of offspring and adaptation to climatic and pollution factors are priority areas. METHODOLOGY/PRINCIPAL FINDINGS: To start to address this situation, expressed genetic transcripts from M. galloprovincialis were pyrosequenced. Transcripts were isolated from the digestive gland, foot, gill and mantle of both male and female mussels. In total, 175,547 sequences were obtained and for foot and mantle, 90% of the sequences could be assembled into contiguous fragments but this reduced to 75% for the digestive gland and gill. Transcripts relating to protein metabolism and respiration dominated including ribosomal proteins, cytochrome oxidases and NADH dehydrogenase subunits. Tissue specific variation was identified in transcripts associated with mitochondrial energy metabolism, with the digestive gland and gill having the greatest transcript abundance. Using fragment recruitment it was also possible to identify sites of potential small RNAs involved in mitochondrial transcriptional regulation. Sex ratios based on Vitelline Envelop Receptor for Lysin and Vitelline Coat Lysin transcript abundances, indicated that an equal sex distribution was maintained. Taxonomic profiling of the M. galloprovincialis tissues highlighted an abundant microbial flora associated with the digestive gland. Profiling of the tissues for genes involved in intermediary metabolism demonstrated that the gill and digestive gland were more similar to each other than to the other two tissues, and specifically the foot transcriptome was most dissimilar. CONCLUSIONS: Pyrosequencing has provided extensive genomic information for M. galloprovincialis and generated novel observations on expression of different tissues, mitochondria and associated microorganisms. It will also facilitate the much needed production of an oligonucleotide microarray for the organism

Adverse Outcome Pathways can drive non-animal approaches for safety assessment

Adverse Outcome Pathways (AOPs) provide an opportunity to develop new and more accurate safety assessment processes for drugs and other chemicals, and may ultimately play an important role in regulatory decision making. Not only can the development and application of AOPs pave the way for the development of improved evidence-based approaches for hazard and risk assessment, there is also the promise of a significant impact on animal welfare, with a reduced reliance on animal-based methods. The establishment of a useable and coherent knowledge framework under which AOPs will be developed and applied has been a first critical step towards realizing this opportunity. This article explores how the development of AOPs under this framework, and their application in practice, could benefit the science and practice of safety assessment, while in parallel stimulating a move away from traditional methods towards an increased acceptance of non-animal approaches. We discuss here the key areas where current, and future initiatives should be focused to enable the translation of AOPs into routine chemical safety assessment, and lasting 3Rs benefits

Impacts of the mycotoxin zearalenone on growth and photosynthetic responses in laboratory populations of freshwater macrophytes (Lemna minor) and microalgae (Pseudokirchneriella subcapitata).

Mycotoxins are an important class of chemicals of emerging concern, recently detected in aquatic environments, potentially reflecting the influence of fungicide resistance and climatic factors on fungal diseases in agricultural crops. Zearalenone (ZON) is a mycotoxin formed by Fusarium spp. and is known for its biological activity in animal tissues; both in vitro and in vivo. ZON has been reported in US and Polish surface waters at 0.7 - 96 ng/L, with agricultural run-off and wastewater treatment plants being the likely sources of mycotoxins. As some mycotoxins can induce phytotoxicity, laboratory studies were conducted to evaluate the toxicity of ZON (as measured concentrations) to freshwater algae (Pseudokirchneriella subcapitata) and macrophytes (Lemna minor) following OECD test guidelines 201 and 221, respectively. Zinc sulphate was used as a positive control. In the OECD 201 algal static study (72 h at 24 ± 1 °C), exposure to ZON gave average specific growth rate (cell density) EC50 and yield (cell density) EC50 values of > 3.1 and 0.92 (0.74 - 1.8) mg/L, respectively. ZON was less toxic in the OECD 221 static study and after 7 d at 24 ± 1 °C. L. minor growth was significantly reduced based on frond number and frond area at 11.4 mg ZON/L, showing a higher tolerance than reported for other mycotoxins with Lemna spp. Chlorophyll fluorescence parameters were used as biomarkers of impacts on photosystem II efficiency, with no effect seen in algae but, with responses being observed in L. minor between 5.2 - 14.4 mg ZON/L. ZON toxicity seen here is not of immediate concern in context with environmental levels, but this study highlights that other freshwater organisms including algae are more sensitive to mycotoxins than Lemna sp., the only current source of toxicity data for freshwater plants

Pathogenic challenge reveals immune trade-off in mussels exposed to reduced seawater pH and increased temperature

Mussels tolerant to seawater pH's that are projected to occur by 2300 due to ocean acidification.•Exposure to pH 6.50 reduced mussel immune response, yet in the absence of a pathogen.•Subsequent pathogenic challenge led to a reversal of immune suppression at pH 6.50.•Study highlights the importance of undertaking multiple stressor exposures.•Shows a need to consider physiological trade-offs and measure responses functionall

Potentially toxic elements status and distribution in Usangu agroecosystem-Tanzania

This study was conducted to highlight status and distribution of potentially toxic elements (PTEs) in Usangu agroecosystem-Tanzania. The study involved 198 soil samples from 10 irrigation schemes including three land use. The concentrations of selected PTEs (Cd, Cr, Cu, Pb, Zn, As, Hg, Co, Nie etc.) were determined to estimate status, distribution, and contamination hierarchy. The total and bioavailable PTEs were determined by aqua regia digestion and Mehlich 3 methods, respectively. We observed variable total PTEs concentration (in mg/kg) among land use and irrigation schemes such as Cr (15), Co (2.9), Fe (7371), Mn (263), and Zn (18.2). Also, concentration of other PTEs (in µg/kg) were As (1382), Cd (22), Cu (3342), Hg (3.3), Ni (4107), and Pb (5661). It was observed that 99.5, 87, 66 and 12% of the studied soils had total Fe, As, Se and Hg concentration above allowable threshold in agricultural soils, respectively. The bioavailable PTEs fraction were lower than total values from different land use and irrigation schemes, higher values of bioavailable PTEs were associated with agricultural intensifications. The status of PTEs in Usangu agroecosystem observed to be at level potentially to cause damaging effect to soil invertebrates, plants, animals and human if remain unregulated. This study highlights baseline information and evidence for site-specific environmental management planning and a scientific basis required to establish PTEs management in agricultural soils to ensure health food and environmental safety