L. Filipe C. Castro

Disruption of zebrafish (Danio rerio) embryonic development after full life-cycle parental exposure to low levels of ethinylestradiol.

Exposure of fish to the synthetic estrogen ethinylestradiol (EE2) has been shown to induce a large set of deleterious effects. In addition to the negative impact of EE2 in reproductive endpoints, concern has recently increased on the potential effects of EE2 in fish embryonic development. Therefore, the present study aimed at examining the effects of EE2 on the full embryonic development of zebrafish in order to identify the actual phases where EE2 disrupts this process. Hence, zebrafish were exposed to environmentally relevant low levels of EE2, 0.5, 1 and 2ng/L (actual concentrations of 0.19, 0.24 and 1ng/L, respectively) from egg up to eight months of age (F(1)), and the survival as well as the occurrence of abnormalities in their offsprings (F(2)), per stage of embryonic development, was investigated. A thorough evaluation of reproductive endpoints and transcription of vtg1 gene in the parental generation (F(1)) at adulthood, was performed. No significant differences could be observed for the two lowest EE2 treatments, in comparison with controls, whereas vtg1 transcripts were significantly elevated (40-fold) in the 2ng/L EE2 treatment. In contrast to the findings in the F(1) generation,a significant concentration-dependent increase in egg mortality between 8 and 24hours post-fertilization (hpf) was observed for all EE2 treatments, when compared with controls. The screening of egg and embryo development showed a significant increase in the percentage of abnormalities at 8 hpf for the highest EE2 concentration, a fact that might explain the increased embryo mortality at the 24 hpf time-point observation. Taken together, these findings indicate that the two lowest tested EE2 concentations impact late gastrulation and/or early organogenesis, whereas exposure to 2ng/L EE2 also disrupts development in the blastula phase. After early organogenesis has been completed (24 hpf), no further mortality was observed. These results show that increased embryo mortality occurs at EE2 levels below those inducing reproductive impairment and vtg1 gene induction in the male parental generation, thus suggesting that EE2 may impact some fish populations at levels below those inducing an increase in vtg1 transcripts. Hence, these findings have important implications for environmental risk assessment, strongly supporting the inclusion of embryonic development studies in the screening of endocrine disruption in wild fish populations.

Long-chain polyunsaturated fatty acid biosynthesis in chordates: Insights into the evolution of Fads and Elovl gene repertoire.

Long-chain polyunsaturated fatty acids (LC-PUFA) are major components of complex lipid molecules and are also involved in numerous critical biological processes. Studies conducted mainly in vertebrates have demonstrated that LC-PUFA can be biosynthesized through the concerted action of two sets of enzymes, namely fatty acyl desaturases (Fads) and elongation of very long-chain fatty acid (Elovl) proteins. While LC-PUFA research is a thriving field, mainly focused on human health, an integrated view regarding the evolution of LC-PUFA biosynthetic genetic machinery in chordates is yet to be produced. Particularly important is to understand whether lineage specific life history trajectories, as well as major biological transitions, or particular genomic processes such as genome duplications have impacted the evolution of LC-PUFA biosynthetic pathways. Here we review the gene repertoire of Fads and Elovl in chordate genomes and the diversity of substrate specificities acquired during evolution. We take advantage of the magnitude of genomic and functional data to show that combination duplication processes and functional plasticity have generated a wide diversity of physiological capacities in extant lineages. A clear evolutionary framework is provided, which will be instrumental for the full clarification of functional capacities between the various vertebrate groups.

The evolutionary history of the stearoyl-CoA desaturase gene family in vertebrates

BackgroundStearoyl-CoA desaturases (SCDs) are key enzymes involved in de novo monounsaturated fatty acid synthesis. They catalyze the desaturation of saturated fatty acyl-CoA substrates at the delta-9 position, generating essential components of phospholipids, triglycerides, cholesterol esters and wax esters. Despite being crucial for interpreting SCDs roles across species, the evolutionary history of the SCD gene family in vertebrates has yet to be elucidated, in particular their isoform diversity, origin and function. This work aims to contribute to this fundamental effort.ResultsWe show here, through comparative genomics and phylogenetics that the SCD gene family underwent an unexpectedly complex history of duplication and loss events. Paralogy analysis hints that SCD1 and SCD5 genes emerged as part of the whole genome duplications (2R) that occurred at the stem of the vertebrate lineage. The SCD1 gene family expanded in rodents with the parallel loss of SCD5 in the Muridae family. The SCD1 gene expansion is also observed in the Lagomorpha although without the SCD5 loss. In the amphibian Xenopus tropicalis we find a single SCD1 gene but not SCD5, though this could be due to genome incompleteness. In the analysed teleost species no SCD5 is found, while the surrounding SCD5-less locus is conserved in comparison to tetrapods. In addition, the teleost SCD1 gene repertoire expanded to two copies as a result of the teleost specific genome duplication (3R). Finally, we describe clear orthologues of SCD1 and SCD5 in the chondrichthian, Scyliorhinus canicula, a representative of the oldest extant jawed vertebrate clade. Expression analysis in S. canicula shows that whilst SCD1 is ubiquitous, SCD5 is mainly expressed in the brain, a pattern which might indicate an evolutionary conserved function.ConclusionWe conclude that the SCD1 and SCD5 genes emerged as part of the 2R genome duplications. We propose that the evolutionary conserved gene expression between distinct lineages underpins the importance of SCD activity in the brain (and probably the pancreas), in a yet to be defined role. We argue that an expression independent of an external stimulus, such as diet induced activity, emerged as a novel function in vertebrate ancestry allocated to the SCD5 isoform in various tissues (e.g. brain and pancreas), and it was selectively maintained throughout vertebrate evolution.

Tributyltin-induced imposex in marine gastropods involves tissue-specific modulation of the retinoid X receptor

Despite the large number of studies on the phenomenon of imposex, the mechanism underlying the abnormal growth of male sexual characters onto females in numerous gastropod species is yet to be fully elucidated. Although several hypotheses have been raised over the years, a convincing body of evidence indicates that tributyltin-induced imposex involves the abnormal modulation of the retinoid X receptor (RXR). Here, we investigate the RXR gene transcription at different timings and tissues upon exposure to environmentally relevant concentrations of tributyltin (TBT) (100 ng Sn/L TBT) in both genders of the imposex susceptible gastropod Nucella lapillus. RXR gene transcription was determined at two time-points (i.e., before and after imposex initiation) by quantitative Real Time PCR in potential target tissues: the central nervous system (CNS), penis/penis forming area (PFA), gonads and digestive gland. TBT-exposure altered transcription of RXR gene in a tissue and sex specific manner. In the CNS, a significant down-regulation was observed in females both before and after imposex initiation (P≤0.01 and P≤0.05, respectively). A similar trend was observed in male CNS at the first time-point, although differences between control and the TBT-exposed group were just above significance (P=0.059). The penis/PFA showed no differences in transcription of RXR gene between control and TBT exposed female snails before imposex induction, or before and after imposex initiation for males. However, male penis showed higher transcription of RXR gene in comparison to the PFA of females. After imposex has been induced, a significant (P≤0.001) increase in transcription of RXR gene was observed in penis of females with vas deference sequence index (VDS) levels of 3-4 in comparison with the PFA of both control and imposex females with VDS 1-2. At advanced stages of imposex, females displayed RXR transcription patterns in penis identical to those of males, which points to a functional role of RXR in the penis of both genders. In the other tissues, gonads and digestive gland, RXR gene transcription was not affected by TBT, at any of the analysed time-points. These patterns of RXR gene transcription upon TBT exposure highlight the pivotal involvement of the CNS in the mechanism of imposex induction. We integrate the results in a conceptual model, and discuss the central role of RXR and the retinoic acid signalling pathways in imposex and male genitalia formation in gastropods.

Is there a compromise between nutrient uptake and gas exchange in the gut of Misgurnus anguillicaudatus, an intestinal air-breathing fish?

The Asian weatherloach, Misgurnus anguillicaudatus (Cobitidae), is a facultative air-breathing teleost fish that makes use of its hindgut or intestine as an accessory air-breathing organ (ABO). The hindgut is highly modified, being well vascularized with intraepithelial capillaries, which makes it well suited for gas exchange. However, the consequences for nutrient uptake, the traditional function of the intestine are unknown. The alimentary canal was examined histologically to assess differences between the fore-, mid- and hindgut regions that have been considered as the digestive, spiral and respiratory zones, respectively. In order to characterise the potential digestive (absorptive) function of the respiratory zone we used semi-quantitative polymerase chain reaction (PCR) to detect the presence of the intestinal Na(+):glucose cotransporter (SGLT1; SLC5A1) and H(+):peptide cotransporter (PEPT1a; SLC15A1) and partially sequenced the SGLT1 and PEPT1a cDNAs. These two transporters play important roles in the absorption of carbohydrate and di-/tripeptides, respectively, in the gut of fishes and other vertebrates and were therefore used as markers for potential nutrient uptake function. We also determined their tissue distributions through semi-quantitative RT-PCR. The effects of diet composition (high protein or high carbohydrate) or fasting on gene expression were also examined. SGLT1 expression was found in kidney, liver, heart, as well as in the three zones of the gut except the most distal part of the hindgut. PEPT1a mRNA was found in heart, brain, liver, and fore- and midgut, but absent in the hindgut. Our results clearly show high expression of SGLT1 (both mRNA and protein by immunolocalization) and PEPT1a (mRNA) in the foregut and midgut correlated with the digestive region of the gut. Modulatory effects of diet on the gene expression for both SGLT1 and PEPT1a were not observed. The presence of SGLT1 transcripts in the respiratory zone of the intestine suggests an overlap in function. However, in the case of PEPT1a, the distal limit was the midgut. Thus, despite its highly modified structure, the hindgut of the loach retains some potential nutrient uptake function.

Organotin levels in seafood from Portuguese markets and the risk for consumers.

Because of their ubiquity in the aquatic environment, the antifouling agent tributyltin (TBT) and other organotins (OTs) accumulate through the food chain, resulting in the occurrence of OTs in seafood products. Despite a high number of studies on the negative impact of TBT in female prosobranch gastropods, few works exist in Europe reporting the levels of these compounds in edible parts of marine organisms used in Human diet. Therefore, within the scope of an EU project OT-SAFE the levels of several OTs were evaluated in the most relevant seafood products for Portuguese consumers. Butyltins (BTs) have been detected in all analysed groups (fish, crustaceans, bivalves, cephalopods), whereas triphenyltin, tricyclohexyltin, monooctyltin and dioctyltin could not be detected and tetrabutyltin was present above detection limits in a single sample. In general, levels of BTs in edible parts of fish, crustaceans and cephalopods collected in Portuguese markets during this study are in the lower range of that reported for these animal groups from other locations (i.e. below 30ngg(-1) wet weight). In contrast, moderate to high concentrations have been observed in bivalves (up to 275ng TBTg(-1) wet weight). While most samples showed TBT plus DBT levels below the tolerable average residue levels (TARL), which may indicate low risk for consumer, four bivalve samples displayed BT levels above TARL, thus indicating that higher bivalve consumer groups may be at risk. The results found are discussed in relation to the potential risk for consumers and integrated with recent finds on the molecular targets of OTs in mammals.

The Mammalian "Obesogen" Tributyltin Targets Hepatic Triglyceride Accumulation and the Transcriptional Regulation of Lipid Metabolism in the Liver and Brain of Zebrafish.

Recent findings indicate that different Endocrine Disrupting Chemicals (EDCs) interfere with lipid metabolic pathways in mammals and promote fat accumulation, a previously unknown site of action for these compounds. The antifoulant and environmental pollutant tributyltin (TBT), which causes imposex in gastropod snails, induces an “obesogenic” phenotype in mammals, through the activation of the nuclear receptors retinoid X receptor (RXR) and peroxisome proliferator-activated receptor gamma (PPARγ). In teleosts, the effects of TBT on the lipid metabolism are poorly understood, particularly following exposure to low, environmental concentrations. In this context, the present work shows that exposure of zebrafish to 10 and 50 ng/L of TBT (as Sn) from pre-hatch to 9 months of age alters the body weight, condition factor, hepatosomatic index and hepatic triglycerides in a gender and dose related manner. Furthermore, TBT modulated the transcription of key lipid regulating factors and enzymes involved in adipogenesis, lipogenesis, glucocorticoid metabolism, growth and development in the brain and liver of exposed fish, revealing sexual dimorphic effects in the latter. Overall, the present study shows that the model mammalian obesogen TBT interferes with triglyceride accumulation and the transcriptional regulation of lipid metabolism in zebrafish and indentifies the brain lipogenic transcription profile of fish as a new target of this compound.

Gene expression analysis of ABC efflux transporters, CYP1A and GSTα in Nile tilapia after exposure to benzo(a)pyrene.

The aim of this study was to evaluate the response of ABC transporters, CYP1A and class alpha (α) GST genes, upon water and dietary exposures to benzo(a)pyrene (BaP) in Oreochromis niloticus. Partial mRNA sequences of ABC transporters (ABCB1b, ABCB11, ABCC1, ABCC2 and ABCG2) were identified, and their tissue distribution patterns evaluated in liver, gill and intestine, showing similarities with other fish and mammals. After 14 days of water exposure to BaP, ABC transporters mRNA expression was up-regulated, namely ABCC2 in gill (up to 16-fold) and ABCG2 in liver (up to 2-fold) and proximal intestine (up to 7-fold). CYP1A mRNA expression was up-regulated in water exposed animals, with maximum fold inductions of 5, 35 and 155, respectively in liver, gill and proximal intestine. After dietary exposure, intestinal CYP1A mRNA showed a 13-fold increase in exposed animals. No significant changes were seen in ABCB1b, ABCC1 and GSTα mRNA expression after both routes of exposure to BaP. In conclusion, this study has shown that transcriptional expression of some ABC transporters and CYP1A respond to the presence of BaP, indicating a possible involvement and cooperation in the detoxification process in Nile tilapia.

A Mollusk Retinoic Acid Receptor (RAR) Ortholog Sheds Light on the Evolution of Ligand Binding

Nuclear receptors are transcription factors that regulate networks of target genes in response to small molecules. There is a strong bias in our knowledge of these receptors because they were mainly characterized in classical model organisms, mostly vertebrates. Therefore, the evolutionary origins of specific ligand-receptor couples still remain elusive. Here we present the identification and characterization of a retinoic acid receptor (RAR) from the mollusk Nucella lapillus (NlRAR). We show that this receptor specifically binds to DNA response elements organized in direct repeats as a heterodimer with retinoid X receptor. Surprisingly, we also find that NlRAR does not bind all-trans retinoic acid or any other retinoid we tested. Furthermore, NlRAR is unable to activate the transcription of reporter genes in response to stimulation by retinoids and to recruit coactivators in the presence of these compounds. Three-dimensional modeling of the ligand-binding domain of NlRAR reveals an overall structure that is similar to vertebrate RARs. However, in the ligand-binding pocket (LBP) of the mollusk receptor, the alteration of several residues interacting with the ligand has apparently led to an overall decrease in the strength of the interaction with the ligand. Accordingly, mutations of NlRAR at key positions within the LBP generate receptors that are responsive to retinoids. Altogether our data suggest that, in mollusks, RAR has lost its affinity for all-trans retinoic acid, highlighting the evolutionary plasticity of its LBP. When put in an evolutionary context, our results reveal new structural and functional features of nuclear receptors validated by millions of years of evolution that were impossible to reveal in model organisms.

Vitellogenin gene expression in the intertidal blenny Lipophrys pholis: a new sentinel species for estrogenic chemical pollution monitoring in the European Atlantic coast?

The presence of estrogenic chemicals (ECs) in the aquatic environment is a growing problem. While most attention was initially given to fresh water and estuarine ecosystems, it is now evident that coastal marine areas are also vulnerable to these pollutants. The use of vitellogenin induction in male fish, a specific biomarker of EC exposure, has been the most widely applied methodology. However, in some occasions, the high mobility and migratory behaviour of common sentinel fish species makes data interpretation difficult. Hence, there is the need to validate new sentinel marine fish species which should display, among other features, a strong homing behaviour. The shanny, Lipophrys pholis, is an intertidal fish that combines many of the required characteristics for a sentinel species: abundance and easy of catch, wide geographical distribution and restricted home range. Thus, in order to evaluate, in the field, the species sensitivity to ECs, L. pholis males were collected at two sites reflecting different degrees of anthropogenic contamination. The vitellogenin II gene (VTGII) was isolated and its liver expression evaluated by RT-PCR in the field samples. A significant induction of gene expression was observed in the specimens collected in the urban area, if compared to the reference site, which suggests exposure to ECs. Moreover, a 21-days laboratory exposure to environmental relevant concentrations of ethinylestradiol (EE2) was also performed. A significant induction of L. pholis VTGII gene in EE2 exposed males was observed suggesting similar sensitivity to that of other marine/estuarine fishes. Even though further validation is currently in progress, the available data indicates that L. pholis is responsive to ECs, thus favouring its future integration in monitoring programmes designed to evaluate the presence of ECs in European marine ecosystems.