Chinese medicine, Qijudihuang pill, mediates cholesterol metabolism and regulates gut microbiota in high-fat diet-fed mice, implications for age-related macular degeneration.

Background: Traditional Chinese Medicines have been used for thousands of years but without any sound empirical basis. One such preparation is the Qijudihuang pill (QP), a mixture of eight herbs, that has been used in China for the treatment of various conditions including age-related macular degeneration (AMD), the most common cause of blindness in the aged population. In order to explain the mechanism behind the effect of QP, we used an AMD model of high-fat diet (HFD) fed mice to investigate cholesterol homeostasis, oxidative stress, inflammation and gut microbiota. Methods: Mice were randomly divided into three groups, one group was fed with control diet (CD), the other two groups were fed with high-fat-diet (HFD). One HFD group was treated with QP, both CD and the other HFD groups were treated with vehicles. Tissue samples were collected after the treatment. Cholesterol levels in retina, retinal pigment epithelium (RPE), liver and serum were determined using a commercial kit. The expression of enzymes involved in cholesterol metabolism, inflammation and oxidative stress was measured with qRT-PCR. Gut microbiota was analyzed using 16S rRNA sequencing. Results: In the majority of the lipid determinations, analytes were elevated by HFD but this was reversed by QP. Cholesterol metabolism including the enzymes of bile acid (BA) formation was suppressed by HFD but again this was reversed by QP. BAs play a major role in signaling between host and microbiome and this is disrupted by HFD resulting in major changes in the composition of colonic bacterial communities. Associated with these changes are predictions of the metabolic pathway complexity and abundance of individual pathways. These concerned substrate breakdowns, energy production and the biosynthesis of pro-inflammatory factors but were changed back to control characteristics by QP. Conclusion: We propose that the ability of QP to reverse these HFD-induced effects is related to mechanisms acting to lower cholesterol level, oxidative stress and inflammation, and to modulate gut microbiota.

In silico analysis of bacterial metabolism of glutamate and GABA in the gut in a rat model of obesity and type 2 diabetes.

Dysbiosis of gut microbiota has adverse effects on host health. This study aimed to determine the effects of changes of faecal microbiota in obese and diabetic rats on the imputed production of enzymes involved in the metabolism of glutamate, gamma-aminobutyric acid (GABA), and succinate. The levels of glutamate decarboxylase, GABA transaminase, succinate-semialdehyde dehydrogenase, and methylisocitrate lyase were reduced or absent in diabetic rats compared with controls and obese rats. Glutamate decarboxylase (GAD) was significantly reduced in obese rats compared with control rats, while the other enzymes were unaltered; different bacterial taxa are suggested to be involved. Levels of bacterial enzymes were inversely correlated with the blood glucose level. These findings suggest that the absence of GABA and reduced succinate metabolism from gut microbiota contribute to the diabetic state in rats.

Characterisation of gut microbiota of obesity and type 2 diabetes in a rodent model.

Various studies have suggested that the gut microbiome interacts with the host and may have a significant role in the aetiology of obesity and Type 2 Diabetes (T2D). It was hypothesised that bacterial communities in obesity and T2D differ from control and compromise normal interactions between host and microbiota. Obesity and T2D were developed in rats by feeding a high-fat diet or a high-fat diet plus a single low-dose streptozotocin administration, respectively. The microbiome profiles and their metabolic potentials were established by metagenomic 16S rRNA sequencing and bioinformatics. Taxonomy and predicted metabolism-related genes in obesity and T2D were markedly different from controls and indeed from each other. Diversity was reduced in T2D but not in Obese rats. Factors likely to compromise host intestinal, barrier integrity were found in Obese and T2D rats including predicted, decreased bacterial butyrate production. Capacity to increase energy extraction via ABC-transporters and carbohydrate metabolism were enhanced in Obese and T2D rats. T2D was characterized by increased proinflammatory molecules. While obesity and T2D show distinct differences, results suggest that in both conditions Bacteroides and Blautia species were increased indicating a possible mechanistic link.

Transcriptome-wide changes associated with the reproductive behaviour of male guppies exposed to 17α-ethinyl estradiol.

Although many pharmaceutical compounds (and their metabolites) can induce harmful impacts at the molecular, physiological and behavioural levels, their underlying mechanistic associations have remained largely unexplored. Here, we utilized RNA-Seq to build a whole brain transcriptome profile to examine the impact of a common endocrine disrupting pharmaceutical (17α-ethinyl estradiol, EE2) on reproductive behaviour in wild guppies (Poecilia reticulata). Specifically, we annotated 16,791 coding transcripts in whole brain tissue in relation to the courtship behaviour (i.e. sigmoid display) of EE2 exposed (at environmentally relevant concentration of 8 ng/L for 28-days) and unexposed guppies. Further, we obtained 10,960 assembled transcripts matching in the non-coding orthologous genomes. Behavioural responses were assessed using a standard mate choice experiment, which allowed us to disentangle chemical cues from visual cues. We found that a high proportion of the RNAseq reads aligned back to our de novo assembled transcriptome with 80.59% mapping rate. Behavioural experiments showed that when males were presented only with female visual cues, there was a significant interaction between male treatment and female treatment in the time spent in the preference zone. This is one of the first studies to show that transcriptome-wide changes are associated with the reproductive behaviour of fish: EE2 exposed male guppies that performed high levels of courtship had a gene profile that deviated the most from the other treatment groups, while both non-courting EE2 and control males had similar gene signatures. Using Gene Ontology pathway analysis, our study shows that EE2-exposed males had gene transcripts enriched for pathways associated with altered immunity, starvation, altered metabolism and spermatogenesis. Our study demonstrates that multiple gene networks orchestrate courting behaviour, emphasizing the importance of investigating impacts of pharmaceuticals on gene networks instead of single genes.

Etifoxine reverses weight gain and alters the colonic bacterial community in a mouse model of obesity.

Obesity is intimately associated with diet and dysbiosis of gut microorganisms but anxiolytics, widely used in treatment of psychiatric conditions, frequently result in weight gain and associated metabolic disorders. We are interested in effects of the anxiolytic etifoxine, which has not been studied with respect to weight gain or effects on gut microorganisms. Here we induced obesity in mice by feeding a high-fat diet but found that intraperitoneal administration of etifoxine resulted in weight loss and decreased serum cholesterol and triglycerides. Obese mice had increased hepatic transcripts associated with lipid metabolism (cyp7a1, cyp27a1, abcg1 and LXRα) and inflammatory factors (TNFα and IL18) but these effects were reversed after etifoxine treatment other than cyp7a1. Taxonomic profiles of the organisms from the caecum were generated by 16S rRNA gene sequencing and Obese and etifoxine mice show differences by diversity metrics, Differential Abundance and functional metagenomics. Organisms in genus Oscillospira and genera from Lachnospiraceae family and Clostridiales order are higher in Control than Obese and at intermediate levels with etifoxine treatment. With respect to community metabolic potential, etifoxine mice have characteristics similar to Control and particularly with respect to metabolism of butanoate, sphingolipid, lipid biosynthesis and xenobiotic metabolism. We suggest mechanisms where-by etifoxine influences processes of host, such as on bile acid synthesis, and microbiota, such as signalling from production of butanoate and sphingosine, resulting in decreased cholesterol, lipids and inflammatory factors. We speculate that the indirect effect of etifoxine on microbial composition is mediated by microbial ß-glucuronidases that metabolise excreted etifoxine glucuronides.

Characterisation of the transcriptome of male and female wild-type guppy brains with RNA-Seq and consequences of exposure to the pharmaceutical pollutant, 17α-ethinyl estradiol.

Waterways are increasingly being contaminated by chemical compounds that can disrupt the endocrinology of organisms. One such compound is 17α-ethinyl estradiol (EE2), a synthetic estrogen used in the contraceptive pill. Despite considerable research interest in the effects of EE2 on reproduction and gene expression, surprisingly, only a few studies have capitalised on technologies, such as next-generation sequencing (NGS), to uncover the molecular pathways related to EE2 exposure. Accordingly, using high-throughput sequencing technologies, the aim of our study was to explore the effects of EE2 on brain transcriptome in wild-type male and female guppy (Poecilia reticulata). We conducted two sets of experiments, where fish were exposed to EE2 (measured concentrations: 8ng/L and 38ng/L) in a flow-through system for 21days. The effects on the brain transcriptome on both males and females were assessed using Illumina sequencing (MiSeq and HiSeq) platform followed by bioinformatics analysis (edgeR, DESeq2). Here, we report that exposure to EE2 caused both up- and downregulation of specific transcript abundances, and affected transcript abundance in a sex-specific manner. Specifically, we found 773 transcripts, of which 60 were male-specific, 61 female-specific and 285 treatment-specific. EE2 affected expression of 165 transcripts in males, with 88 downregulated and 77 upregulated, while in females, 120 transcripts were affected with 62 downregulated and 58 upregulated. Finally, RT-qPCR validation demonstrated that expression of transcripts related to transposable elements, neuroserpin and heat shock protein were significantly affected by EE2-exposure. Our study is the first to report brain transcriptome libraries for guppies exposed to EE2. Not only does our study provide a valuable resource, it offers insights into the mechanisms underlying the feminizing effects on the brains of organisms exposed to environmentally realistic concentrations of EE2.

Histological Characterization of the Dicer1 Mutant Zebrafish Retina.

DICER1, a multidomain RNase III endoribonuclease, plays a critical role in microRNA (miRNA) and RNA-interference (RNAi) functional pathways. Loss of Dicer1 affects different developmental processes. Dicer1 is essential for retinal development and maintenance. DICER1 was recently shown to have another function of silencing the toxicity of Alu RNAs in retinal pigment epithelium (RPE) cells, which are involved in the pathogenesis of age related macular degeneration. In this study, we characterized a Dicer1 mutant fish line, which carries a nonsense mutation (W1457Ter) induced by N-ethyl-N-nitrosourea mutagenesis. Zebrafish DICER1 protein is highly conserved in the evolution. Zebrafish Dicer1 is expressed at the earliest stages of zebrafish development and persists into late developmental stages; it is widely expressed in adult tissues. Homozygous Dicer1 mutant fish (DICER1(W1457Ter/W1457Ter)) have an arrest in early growth with significantly smaller eyes and are dead at 14-18 dpf. Heterozygous Dicer1 mutant fish have similar retinal structure to that of control fish; the retinal pigment epithelium (RPE) cells are normal with no sign of degeneration at the age of 20 months.

Key metabolic pathways involved in xenobiotic biotransformation and stress responses revealed by transcriptomics of the mangrove oyster Crassostrea brasiliana.

The Brazilian oyster Crassostrea brasiliana was challenged to three common environmental contaminants: phenanthrene, diesel fuel water-accommodated fraction (WAF) and domestic sewage. Total RNA was extracted from the gill and digestive gland, and cDNA libraries were sequenced using the 454 FLX platform. The assembled transcriptome resulted in ̃20,000 contigs, which were annotated to produce the first de novo transcriptome for C. brasiliana. Sequences were screened to identify genes potentially involved in the biotransformation of xenobiotics and associated antioxidant defence mechanisms. These gene families included those of the cytochrome P450 (CYP450), 70kDa heat shock, antioxidants, such as glutathione S-transferase, superoxide dismutase, catalase and also multi-drug resistance proteins. Analysis showed that the massive expansion of the CYP450 and HSP70 family due to gene duplication identified in the Crassostrea gigas genome also occurred in C. brasiliana, suggesting these processes form the base of the Crassostrea lineage. Preliminary expression analyses revealed several candidates biomarker genes that were up-regulated during each of the three treatments, suggesting the potential for environmental monitoring.

A light in the darkness: new biotransformation genes, antioxidant parameters and tissue-specific responses in oysters exposed to phenanthrene.

Phenanthrene (PHE), a major component of crude oil, is one of the most abundant polycyclic aromatic hydrocarbons (PAHs) in aquatic ecosystems, and is readily bioavailable to marine organisms. Understanding the toxicity of PAHs in animals requires knowledge of the systems for xenobiotic biotransformation and antioxidant defence and these are poorly understood in bivalves. We report, for the first time, new transcripts and tissue-specific transcription in gill and digestive gland from the oyster Crassostrea brasiliana following 24h exposure to 100 and 1000µgL(-1) PHE, a model PAH. Six new cytochrome P450 (CYP) and four new glutathione S-transferase (GST) genes were analysed by means of quantitative reverse transcription PCR (qRT-PCR). Different antioxidant endpoints, including both enzymatic and non-enzymatic parameters, were assessed as potential biomarkers of oxidative stress. GST activity was measured as an indicator of phase II biotransformation. Rapid clearance of PHE was associated with upregulation of both phase I and II genes, with more pronounced effects in the gill at 1000µgL(-1) PHE. After 24h of exposure, PHE also caused impairment of the antioxidant system, decreasing non-protein thiols and glutathione levels. On the other hand, no change in antioxidant enzymes was observed. PHE treatment (100µgL(-1)) significantly decreased GST activity in the gill of exposed oysters. Both CYP and GST were transcribed in a tissue-specific manner, reflecting the importance of the gill in the detoxification of PAHs. Likewise, the antioxidant parameters followed a similar pattern. The data provide strong evidence that these genes play key roles in C. brasiliana biotransformation of PHE and highlight the importance of gill in xenobiotic metabolism.

Effects of 17α-ethinyl estradiol exposure on estrogen receptors α and ß and vitellogenins A, B and C mRNA expression in the liver of sand goby (Pomatoschistus minutus).

This study aims to characterize the estrogen receptor (er) in sand goby (Pomatoschistus minutus) and determine the temporal effects of 17α-ethinyl estradiol (EE2) on erα and vitellogenin (vtg) gene expression in males. Two partial cDNA sequences (erα and erß1) are presented showing conserved structural features with ers of other species. Transcript levels for both ers were low in control fish but EE2 exposure (11 ng/L, for 29 days) increased both to a pattern similar to vitellogenic females. The relative expression of three vtg genes (vtga, vtgb and vtgc) along with erα was determined in control and male fish exposed to EE2 (11 ng/L) at multiple time-points over 29 days. All four transcripts were significantly induced due to exposure and expression rose during the time course with distinct temporal patterns and vtga reached a substantially higher level at the end of the time course coinciding with rapid elevation in erα expression.

Characterisation of genes transcriptionally upregulated in the liver of sand goby (Pomatoschistus minutus) by 17α-ethinyloestradiol: identification of distinct vitellogenin and zona radiata protein transcripts.

The sand goby (Pomatoschistus minutus), is a marine and estuarine teleost that is used in environmental, reproductive and behavioural studies of oestrogenic endocrine disruption. The xeno-oestrogen, 17α-ethinyloestradiol (EE2), induces expression of egg proteins vitellogenin (VTG) and zona radiata protein (ZRP) in male fish and impairs reproduction. Multiple forms of VTG and ZRP genes are found in other teleost species, yet the characteristics of VTG and ZRP in the sand goby are unknown. In this investigation, Suppressive Subtractive Hybridization was used to isolate cDNA fragments from liver, identified as belonging to 11 distinct sand goby genes, suggesting that these genes are transcriptionally upregulated by EE2. Assembly of these fragments revealed three VTG genes which shared homology with VTG classes A, B and C in other fish and two ZRP genes sharing homology with ZRP classes Ba and Bb. RTqPCR of RNA from the sand goby liver was used to show that these VTGs and ZRPs were present in low levels in control males and high levels in mature females. Exposure of males to a concentration of 11 ngL(-1) EE2 caused a significant increase in all VTG and ZRP transcript levels. The identification of these egg protein transcripts and the development of validated assays for their quantification will facilitate future work with this useful model species.

Annual variation in the levels of transcripts of sex-specific genes in the mantle of the common mussel, Mytilus edulis.

Mytilus species are used as sentinels for the assessment of environmental health but sex or stage in the reproduction cycle is rarely considered even though both parameters are likely to influence responses to pollution. We have validated the use of a qPCR assay for sex identification and related the levels of transcripts to the reproductive cycle. A temporal study of mantle of Mytilus edulis found transcripts of male-specific vitelline coat lysin (VCL) and female-specific vitelline envelope receptor for lysin (VERL) could identify sex over a complete year. The levels of VCL/VERL were proportional to the numbers of sperm/ova and are indicative of the stage of the reproductive cycle. Maximal levels of VCL and VERL were found in February 2009 declining to minima between July - August before increasing and re-attaining a peak in February 2010. Water temperature may influence these transitions since they coincide with minimal water temperature in February and maximal temperature in August. An identical pattern of variation was found for a cryptic female-specific transcript (H5) but a very different pattern was observed for oestrogen receptor 2 (ER2). ER2 varied in a sex-specific way with male > female for most of the cycle, with a female maxima in July and a male maxima in December. Using artificially spawned animals, the transcripts for VCL, VERL and H5 were shown to be present in gametes and thus their disappearance from mantle is indicative of spawning. VCL and VERL are present at equivalent levels in February and July-August but during gametogenesis (August to January) and spawning (March to June) VCL is present at lower relative amounts than VERL. This may indicate sex-specific control mechanisms for these processes and highlight a potential pressure point leading to reduced reproductive output if environmental factors cause asynchrony to gamete maturation or release.

Exposure to 17alpha-ethinyl estradiol impairs courtship and aggressive behaviour of male sand gobies (Pomatoschistus minutus).

Endocrine disrupting chemicals (EDCs), natural or manmade, are known to interfere with the endocrinology of organisms and also cause behavioural changes. The aim of this study was to test how 1-4weeks exposure to 17alpha-ethinyl estradiol (EE2, 11ngL(-1)) affects nest building, courtship and aggressive behaviour of male fish. Our study species, the sand goby (Pomatoschistus minutus) exhibits a polygynous mating system, in which males compete for females and defend their nest against intruders. Nest takeovers are common in their nest-constrained habitat. In our experiment, control and EE2-exposed males were first allowed to build a nest and mate with non-exposed females. When the males had received eggs in their nest, three rival males were introduced into the test aquarium, and the males were left to compete for the nest site overnight. Courtship and aggressive defence behaviour were recorded using a video camera. In addition to behavioural endpoints we measured the expression of hepatic vitellogenin (Vtg) and zona radiata protein (Zrp) mRNA, and several common somatic indices. Our study showed that exposure to EE2 delays nest building and decreases male courtship and leading behaviour. Additionally, EE2-exposed males were significantly less aggressive than control males. Nest takeover rate was not affected by EE2 exposure: an equal percent of males in both treatments lost their nest to competitors. EE2 exposure also induced Vtg and Zrp mRNA expression in males and decreased the hepatosomatic index. The observed changes in nest building, courtship and aggressive behaviour of EE2-exposed males are likely to have negative implications for male reproductive success.

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.

Integrating omic technologies into aquatic ecological risk assessment and environmental monitoring: hurdles, achievements, and future outlook.

BACKGROUND: In this commentary we present the findings from an international consortium on fish toxicogenomics sponsored by the U.K. Natural Environment Research Council (Fish Toxicogenomics-Moving into Regulation and Monitoring, held 21-23 April 2008 at the Pacific Environmental Science Centre, Vancouver, BC, Canada). OBJECTIVES: The consortium from government agencies, academia, and industry addressed three topics: progress in ecotoxicogenomics, regulatory perspectives on roadblocks for practical implementation of toxicogenomics into risk assessment, and dealing with variability in data sets. DISCUSSION: Participants noted that examples of successful application of omic technologies have been identified, but critical studies are needed to relate molecular changes to ecological adverse outcome. Participants made recommendations for the management of technical and biological variation. They also stressed the need for enhanced interdisciplinary training and communication as well as considerable investment into the generation and curation of appropriate reference omic data. CONCLUSIONS: The participants concluded that, although there are hurdles to pass on the road to regulatory acceptance, omics technologies are already useful for elucidating modes of action of toxicants and can contribute to the risk assessment process as part of a weight-of-evidence approach.

An endocrine disrupting chemical changes courtship and parental care in the sand goby.

Endocrine disrupting chemicals (EDCs) are a diverse group of compounds that can mimic, block or modulate the synthesis of natural hormones. They are known to cause impairment of reproduction of aquatic organisms at very low concentrations. The aim of this study was to examine how exposure from 10 to 31 days to 17alpha-ethinyl estradiol (EE2, 41 ng L(-1)) affects the courtship and parental care behaviour of male sand gobies (Pomatoschistus minutus). The sand goby exhibits a polygynous mating system, where males compete for females and provide paternal care. First, male courtship performance towards a stimulus female was recorded with video camera. Secondly, after the male had received eggs his parental care behaviour was video recorded. In addition to behavioural endpoints, we measured the expression of hepatic vitellogenin (Vtg) and zona radiata protein (Zrp) mRNA, as well as common somatic indices. Our study shows that exposure to EE2 affected male fanning behaviour during both courtship and parental care. Interestingly, small exposed males increased their courtship fanning to similar levels as larger control males. However, during parental care egg fanning was not related to male size, and all exposed males fanned more than control males. The EE2-exposure induced Vtg and Zrp mRNA expression in males and decreased hepatosomatic index (HSI), and increased gonadosomatic index (GSI). Females prefer males that fan more, which will favour the small EDC exposed males. This may lead to mating that favours males that are not strong enough to tend the eggs until they hatch, thus decreasing the reproductive success of individuals.

Hepatic transcriptomic and metabolomic responses in the Stickleback (Gasterosteus aculeatus) exposed to ethinyl-estradiol.

An established three-spined stickleback (Gasterosteus aculeatus) cDNA array was expanded to 14,496 probes with the addition of hepatic clones derived from subtractive and normalized libraries from control males and males exposed to model toxicants. Microarrays and one-dimensional (1)H nuclear magnetic resonance (NMR) spectroscopy, together with individual protein and gene biomarkers were employed to investigate the hepatic responses of the stickleback to ethinyl-estradiol (EE(2)) exposure. Male fish were exposed via the water to EE(2), including environmentally relevant concentrations (0.1-100ng/l) for 4 days, and hepatic transcript and metabolite profiles, kidney spiggin protein and serum vitellogenin concentrations were determined in comparison to controls. EE(2) exposure did not significantly affect spiggin concentration but significantly induced serum vitellogenin protein at the threshold concentration of 32ng/l. (1)H NMR coupled with robust univariate testing revealed only limited changes, but these did support the predicted modulation of the amino acid profile by transcriptomics. Transcriptional induction was found for hepatic vitellogenins and choriogenins as expected, together with a range of other EE(2)-responsive genes. Choriogenins showed the more sensitive responses with statistically significant induction at 10ng/l. Real-time polymerase chain reaction (PCR) confirmed transcriptional induction of these genes. Phosvitinless vitellogenin C transcripts were highly expressed and represent a major form of the egg yolk precursors, and this is in contrast to other fish species where it is a minor component of vitellogenic transcripts. Differences in inducibility between the vitellogenins and choriogenins appear to be in accordance with the sequential formation of chorion and yolk during oogenesis in fish.

Differential expression of vitellogenin and oestrogen receptor genes in the liver of zebrafish, Danio rerio.

Environmental oestrogens are responsible for adverse effects in fish that affect reproduction. Availability of model fish to study the differential effects of endogenous and exogenous oestrogens and to test for oestrogenic activity of chemicals would be advantageous. Zebrafish could provide such a model, but the organisation and expression of vitellogenins (VTGs) and oestrogen receptors (ERs) are not completely understood. VTGs are synthesised in the liver and provide a sensitive biomarker of oestrogenic activity since they are thought to be under the regulation of the ER. There are multiple genes for VTGs and an in silico analysis of their distribution in the Zebrafish genome has identified six genes: VTG-1, VTG-2, VTG-4, VTG-5, VTG-7 located on chromosome 22 and VTG-3 on chromosome 11. VTG-specific, quantitative, real-time, reverse-transcriptase polymerase chain reaction assays were developed and used to measure differential expression in the livers of mature male and female zebrafish. Following normalisation in female fish, relative expression of VTG-5 mRNA is highest and is 1.3x, 1.6x and 2x higher than VTG-4, VTG-2 and VTG-1, respectively, while expression of VTG-3 and VTG-7 is very low. Expression of VTGs in male fish was either undetectable or very low (VTG-4 and VTG-5). ERalpha and ERbeta2 were expressed at higher levels than ERbeta1 in females, but only ERbeta2 was expressed in appreciable quantity in males. Expression of ERalpha in males was significant but only at the limit of detection (<0.1% of female fish), while ERbeta1 could not be detected. The very low level of expression of ERalpha in males raises questions about the accepted mechanism of oestrogenic induction of VTG in male fish.

Hepatic transcriptomic and metabolomic responses in the stickleback (Gasterosteus aculeatus) exposed to environmentally relevant concentrations of dibenzanthracene.

A three-spined stickleback (Gasterosteus aculeatus) cDNA array and one-dimensional 1H nuclear magnetic resonance (NMR) spectroscopy-based metabolomics approach, together with individual biomarkers,were employed to investigate the responses of male sticklebacks to polycyclic aromatic hydrocarbon exposure. Fish were exposed to 1,2:5,6-dibenzanthracene (DbA) at concentrations between 0.01 and 50 microg per liter dissolved in the ambient water for four days, and hepatic transcript and metabolite profiles were determined in comparison with those of solvent-exposed controls. Induction of gene expression was apparent for cytochrome P450 1A (CYP1A) and CYP2-family monooxygenases and these responses were strongly correlated with DbA exposure concentrations (for CYP1A r > 0.996). Expression of suites of genes related to bile acid biosynthesis, steroid metabolism, and endocrine function were also affected, as demonstrated by gene ontology analyses. Expression changes in selected genes were confirmed by real-time PCR. Metabolomics highlighted notable changes in concentrations of taurine, malonate, glutamate, and alanine. These statistically significant responses to environmentally relevant concentrations of DbA at the transcriptomic and metabolomic levels provided sensitive markers characteristic of environmentally relevant low-level DbA exposure. Metabolic pathways were identified where both gene expression and metabolite concentrations were altered in response to DbA.

Sand goby (Pomatoschistus minutus) males exposed to an endocrine disrupting chemical fail in nest and mate competition.

Endocrine disrupting chemicals (EDCs) are a widely studied group of chemicals that interfere with the endocrinology of organisms. So far, few studies have demonstrated the effect of EDCs on the reproductive behavior of aquatic wildlife. Here we show that sand goby males' (Pomatoschistus minutus) success in mating competition greatly decreases after an exposure for 7 to 24 days to 17alpha-ethinyl estradiol (EE2, measured concentration 4 ng L(-1)). The sand goby exhibits a polygynous mating system with male parental care, in which males compete for nest sites and females. The aim of this study was to test how EE2 exposure affects the ability of males to compete for breeding resources, i.e. nest sites and mates. First, EE2 exposed males competed over a nest site against a non-exposed, control male of the same size. Secondly, we examined male courtship behavior and female mate preferences for EE2 exposed males and similar-sized non-exposed, control males. In addition to the behavioral experiments we determined the zona radiata protein (Zrp) mRNA gene expression and measured morphometric indicators of sexual maturation. Our study revealed that EE2 treated males were not able to acquire or defend a nest site. Additionally, EE2 treated males spent significantly less time in active courtship and nest leading behavior than control males. As a result, females clearly preferred to mate with control males. However, we found no significant differences in Zrp mRNA expression or the morphometric indicators between treatments. Our study illustrates that exposure to this EDC can greatly reduce the chances of an individual reproducing successfully. Moreover, it demonstrates that severe behavioral effects can be seen before any effects are detectable at the molecular or morphometric level.