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

Unravelling the neurophysiological basis of aggression in a fish model

<p>Abstract</p> <p>Background</p> <p>Aggression is a near-universal behaviour with substantial influence on and implications for human and animal social systems. The neurophysiological basis of aggression is, however, poorly understood in all species and approaches adopted to study this complex behaviour have often been oversimplified. We applied targeted expression profiling on 40 genes, spanning eight neurological pathways and in four distinct regions of the brain, in combination with behavioural observations and pharmacological manipulations, to screen for regulatory pathways of aggression in the zebrafish (<it>Danio rerio</it>), an animal model in which social rank and aggressiveness tightly correlate.</p> <p>Results</p> <p>Substantial differences occurred in gene expression profiles between dominant and subordinate males associated with phenotypic differences in aggressiveness and, for the chosen gene set, they occurred mainly in the hypothalamus and telencephalon. The patterns of differentially-expressed genes implied multifactorial control of aggression in zebrafish, including the hypothalamo-neurohypophysial-system, serotonin, somatostatin, dopamine, hypothalamo-pituitary-interrenal, hypothalamo-pituitary-gonadal and histamine pathways, and the latter is a novel finding outside mammals. Pharmacological manipulations of various nodes within the hypothalamo-neurohypophysial-system and serotonin pathways supported their functional involvement. We also observed differences in expression profiles in the brains of dominant versus subordinate females that suggested sex-conserved control of aggression. For example, in the HNS pathway, the gene encoding arginine vasotocin (AVT), previously believed specific to male behaviours, was amongst those genes most associated with aggression, and AVT inhibited dominant female aggression, as in males. However, sex-specific differences in the expression profiles also occurred, including differences in aggression-associated tryptophan hydroxylases and estrogen receptors.</p> <p>Conclusions</p> <p>Thus, through an integrated approach, combining gene expression profiling, behavioural analyses, and pharmacological manipulations, we identified candidate genes and pathways that appear to play significant roles in regulating aggression in fish. Many of these are novel for non-mammalian systems. We further present a validated system for advancing our understanding of the mechanistic underpinnings of complex behaviours using a fish model.</p

Health Impacts of Estrogens in the Environment, Considering Complex Mixture Effects

PublishedResearch Support, Non-U.S. Gov'tBACKGROUND: Environmental estrogens in wastewater treatment work (WwTW) effluents are well established as the principal cause of reproductive disruption in wild fish populations, but their possible role in the wider health effects of effluents has not been established. OBJECTIVES: We assessed the contribution of estrogens to adverse health effects induced in a model fish species by exposure to WwTW effluents and compared effects of an estrogen alone and as part of a complex mixture (i.e., spiked into effluent). METHODS: Growth, genotoxic, immunotoxic, metabolic, and endocrine (feminized) responses were compared in fathead minnows (Pimephales promelas) exposed for 21 days to a potent estrogenic effluent, a weakly estrogenic effluent before and after spiking with a steroidal estrogen [17 alpha-ethinyl-estradiol (EE2)], and to EE2 alone. RESULTS: In addition to endocrine disruption, effluent exposure induced genotoxic damage, modulated immune function, and altered metabolism; many of these effects were elicited in a sex-specific manner and were proportional to the estrogenic potencies of the effluents. A key finding was that some of the responses to EE2 were modified when it was present in a complex mixture (i.e., spiked into effluent), suggesting that mixture effects may not be easily modeled for effluent discharges or when the chemicals impact on a diverse array of biological axes. CONCLUSION: These data reveal a clear link between estrogens present in effluents and diverse, adverse, and sex-related health impacts. Our findings also highlight the need for an improved understanding of interactive effects of chemical toxicants on biological systems for understanding health effects of environmental mixtures.This work was funded by the Environment Agency to C.R.T. (project no. SC040078). T.N. was supported by a Fundação para a Ciência ea Tecnologia (FCT) fellowship (BPD/18192/04)

Introduction to special issue:New Times Revisited: Britain in the 1980s

The authors in this volume are collectively engaged with a historical puzzle: What happens if we examine the decade once we step out of the shadows cast by Thatcher? That is, does the decade of the 1980s as a significant and meaningful periodisation (equivalent to that of the 1960s) still work if Thatcher becomes but one part of the story rather than the story itself? The essays in this collection suggest that the 1980s only makes sense as a political period. They situate the 1980s within various longer term trajectories that show the events of the decade to be as much the consequence as the cause of bigger, long-term historical processes. This introduction contextualises the collection within the wider literature, before explaining the collective and individual contributions made