D Lima

University of Porto, Oporto, Porto, Portugal

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Publications (14)41.79 Total impact

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    ABSTRACT: Nuclear receptors (NRs) 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, since 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 RXR. Surprisingly, we also find that NlRAR does not bind all-trans retinoic acid (RA) 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 co-activators in the presence of these compounds. 3D 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 RA, highlighting the evolutionary plasticity of its LBP. When put in an evolutionary context, our results reveal new structural and functional features of NRs validated by millions of years of evolution that were impossible to reveal in model organisms.
    Endocrinology 08/2014; · 4.72 Impact Factor
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    ABSTRACT: HSD17B12 is a member of the hydroxysteroid dehydrogenase superfamily, a multifunctional group of enzymes involved in the metabolism of steroids, retinoids, bile and fatty acids. Whether the main role of HSD17B12 in mammals is in steroid or fatty acid metabolism is a subject of intense debate. In mollusks it has been shown that an HSD17B12 orthologue can convert estrone into estradiol in vitro, although its primary in vivo function remains unknown. To gain insight into its role in gastropods, we provide here the first cloning of Hsd17b12 in Nucella lapillus and its detailed tissue distribution through quantitative PCR. Furthermore, given that the endocrine disruptor tributyltin (TBT) has been reported to unbalance steroid and lipid levels in gastropods, we tested its impact in on NlHsd17b12 transcript expression. Our results show that NlHsd17b12 is ubiquitously expressed in all tissues analyzed, with higher levels in organs with high metabolic rates, such as kidney and digestive gland, a pattern consistent with an involvement in lipid metabolism. Exposure to TBT chloride at 100ng Sn/L caused a decrease in NlHsd17b12 mRNA levels in digestive gland, after one and two months, while no effect was observed in gonads. Overall, these results suggest that in mollusks, as in mammals, this enzyme is likely to be involved in lipid metabolism, and emphasize the need to perform more detailed studies on its in vivo function, in order to understand its physiological role and the biological impact of its disruption by pollutants such as TBT.
    The Journal of steroid biochemistry and molecular biology 10/2012; · 3.98 Impact Factor
  • Journal of Molluscan Studies 09/2012; · 1.36 Impact Factor
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    ABSTRACT: Oil spills are an importance source of polycyclic aromatic hydrocarbons (PAHs) in the aquatic environment. Intertidal communities are particularly sensitive since most organisms from these ecosystems are sessile or present reduced mobility. Hence, it is important to validate the use of resident species as sentinels to characterize the impact of oil spills on the rocky shores and the improvement during the restoration process. Recently the advantages of using the shanny Lipophrys pholis in pollution monitoring within the northwestern Atlantic coast has been pointed out. Therefore, with the aim of further validating the use of L. pholis in pollution monitoring associated with petrogenic hydrocarbon contamination, a multi-biomarker approach study was carried out 1 week after a moderate oil spill from the waste treatment plant (WTP) of the major Portuguese refinery in the north of Portugal (Petrogal). Fish collected at 2 km from the accident displayed a significant induction of ethoxyresorufin-O-deethylase activity (EROD) and fluorescent aromatic compounds (FACs) in bile (up to a 5-fold induction) in comparison with the pre-spill scenario, and a 15% induction in erythrocytic nuclear abnormalities (ENA), a biomarker of genotoxicity. In contrast, no significant differences were recorded in the reference site. In order to better characterize the time-course accumulation of FACs in bile after a PAH insult, laboratory exposure of L. pholis to benzo[a]pyrene (B[a]P) was performed. A clear dose-response accumulation of B[a]P metabolites was observed that closely reflected nominal exposure concentrations already after 3d. Overall, the findings of the present study highlight the potential of L. pholis in pollution monitoring dealing not only with chronic contamination, but also with oil spill accidents of a moderate scale. Taking into consideration that EROD and FACs determinations in L. pholis are cost effective, rapid and easy to use, they offer a great potential to be incorporated into risk assessment of PAHs in the scope of national monitoring programs and the European Water Policy legislation.
    Chemosphere 10/2010; 81(6):685-91. · 3.14 Impact Factor
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    ABSTRACT: 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.
    Aquatic toxicology (Amsterdam, Netherlands) 10/2010; 101(1):221-7. · 3.12 Impact Factor
  • Comparative Biochemistry and Physiology Part C Toxicology & Pharmacology 02/2009; 149:58-64. · 2.71 Impact Factor
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    ABSTRACT: Recent studies performed under laboratory conditions have shown that single exposure to high levels of several xenoestrogens is able to induce imposex in at least two neogastropod species. In an attempt to evaluate if xenoestrogens, at environmentally relevant conditions, do contribute to imposex induction, we have tested the effects of a mixture containing xenoestrogens (municipal sewage effluents) on imposex development in the dogwhelk Nucella lapillus. Exposure for 3 months to the raw (0.25% and 1%) and the final sewage effluent (12.5% and 50%) rendered no increase in the severity of imposex. Conversely, as exposure to high concentrations of natural steroids, estradiol and estrone, had previously been shown to partially rescue imposex development under laboratory conditions, we have also tested if exposure to the final sewage effluent could ameliorated the severity of imposex induction by tributyltin (TBT). The results demonstrated that co-exposure to the final sewage effluent leads to a decrease trend in the severity of imposex in the presence of TBT. Within the studied imposex parameters, the Relative Penis Size index (RPSI) was the most affected with a 50% decrease in the effluent 12.5% plus TBT exposed group and 25% decrease in the effluent 50% plus TBT, if compared with the TBT alone. Overall, our results give further support to the use of imposex in N. lapillus as a specific biomarker of TBT contamination. However, in areas of high inputs of sewage effluents, the assessment of TBT contamination by the use of the imposex phenomenon should ideally also include data on the tissue levels of butyltins.
    Comparative Biochemistry and Physiology Part C Toxicology & Pharmacology 08/2008; 148(1):87-93. · 2.71 Impact Factor
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    ABSTRACT: 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.
    Comparative Biochemistry and Physiology Part C Toxicology & Pharmacology 08/2008; 149(1):58-64. · 2.71 Impact Factor
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    ABSTRACT: This study is part of a project aiming to validate the use of the intertidal shanny Lipophorys pholis as a sentinel species in pollution monitoring in NW European marine ecosystems. To this end, a characterisation of acethylcholin (AChE), butyrylcholin (BChE) and propionylcholin (PrChE) esterases in L. pholis muscle was performed and the results indicated that AChE was predominant. Furthermore, the use of eserine sulphate and BW284c51 (0.64-800 microM), and iso-OMPA (0.08-16 mM), confirmed the measurement of true cholinesterases (ChEs) as well as the presence of pseudocholinesterases. The field application of these markers to L. pholis, sampled in seven locations along the Portuguese coast, revealed that fish were likely to be affected by neurotoxic compounds. This was indicated by the significant depletion of AChE (p<0.05) in animals collected at urban and industrialised sites, compared with those from reference locations. The inclusion of a marker of effect, measured as lipid peroxidation levels in muscle tissue, also revealed the existence of site differences. Overall, the study further validates the utility of L. pholis in pollution monitoring studies.
    Marine Pollution Bulletin 05/2008; 56(5):999-1007. · 2.53 Impact Factor
  • Bulletin of Environmental Contamination and Toxicology 02/2008; 80(1):19-23. · 1.11 Impact Factor
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    ABSTRACT: The contamination of aquatic ecosystems by organic contaminants such as polycyclic aromatic hydrocarbons (PAHs) is a matter of great concern. Mussels have been extensively used as sentinel species in a large number of monitoring programs. However, the use of bivalves as the sole species has some limitations, because they are not as responsive as fish to Aryl Hydrocarbon Receptor agonists. Hence, for many marine areas, there is the need to validate new sentinel fish species that can be used in the assessment of pollution by organic contaminants. The shanny Lipophrys pholis is an intertidal fish that combines many characteristics required in a sentinel species: is abundant and easy to catch, has a wide geographical distribution and restrict home range. After larvae recruitment to the intertidal rocky shores, they remain in the same area for the rest of the life-cycle, thus reflecting local pollutants exposure. In order to evaluate the species sensitivity to organic contaminants under field conditions, L. pholis were collected at six sites reflecting different degrees of anthropogenic contamination. The induction of two biomarkers extensively validated in the assessment of PAHs contamination ethoxyresorufin-O-deethylase activity (EROD) and Fluorescent Aromatic Compounds (FACs) was evaluated. In parallel, mussels were collected at the same locations and levels of 16 PAHs and selected heavy metals determined. Overall, the specimens collected in the urban areas showed a significant induction of EROD and FACs (up to a six-fold induction) if compared with the reference sites. Additionally, a positive correlation was observed between the biomarkers and PAHs levels in mussel tissues. Even though further validation is currently in progress, the available data indicate that L. pholis is responsive to organic contaminants such as PAHs, suggesting its future integration in monitoring programmes designed to evaluate the presence of these contaminants in European marine ecosystems.
    Environment International 02/2008; 34(1):94-101. · 6.25 Impact Factor
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    ABSTRACT: The imposex phenomenon in female prosobranch gastropods provides one of the best documented examples of endocrine disruption in wildlife. While many field studies have demonstrated the negative impact of tributyltin (TBT) upon female gastropods, the mechanism(s) underlying imposex development has not yet been fully clarified. Over the years several hypotheses have been raised to determine the biochemical and molecular determinants of this process. Nevertheless, the interplay between the different suggested pathways (neuroendocrine, steroid and retinoid) is still unknown. Hence, through a combination of exposure experiments, we show that the 9-cis-retinoic acid (9cisRA), the proposed natural ligand of the retinoic X receptor (RXR), induces imposex in females of Nucella lapillus to the same degree as tributyltin, when administered at similar concentrations (1 microg/g body weight). Methoprene acid, a selective ligand for RXR, also induces imposex, albeit to a lower degree than that of the positive control. In contrast, testosterone significantly induced imposex, but had no effect on female penis induction, while the neuropeptide APGWamide had no effect on imposex development. These results clearly demonstrate that imposex induction in N. lapillus is mediated through the modulation of the RXR signalling pathways. In addition to the effects reported in female dogwhelks, both TBT and RA significantly increased male penis length, thus suggesting that TBT may also impact male secondary sex organs through the RXR signalling pathways. As a step for future studies, we have cloned the orthologue of N. lapillus RXR and provide experimental evidence that it binds 9cisRA. Finally, the basal expression level of RXR in several tissues of N. lapillus was determined through real-time PCR, thus showing that RXR is ubiquitously expressed in mollusc tissues, with the highest expression levels being recorded in female and male gonads. The mechanistic impacts of the overall findings to the imposex process are discussed.
    Aquatic Toxicology 12/2007; 85(1):57-66. · 3.73 Impact Factor
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    ABSTRACT: The molecular targets of estrogenic endocrine disrupting chemicals have been studied in detail in vertebrates. The lack of basic endocrine knowledge impairs similar approaches for invertebrates. Evidence indicates that the signalling pathways of invertebrates may also be a target of estrogenic chemicals (ECs). In fact, the exposure to effluents containing ECs has been reported to impact mollusc reproduction. Despite the reported estrogen independence of the mollusc nuclear estrogen receptor (ER), its role in EC-induced toxicity has not been investigated in vivo. Therefore, we have cloned the ER of the gastropod Nucella lapillus and evaluated the effects of a mixture of estrogenic chemicals (sewage effluent) on its expression in the ovary. Here, we show that the exposure to a raw domestic/industrial effluent, impact ER expression with a simultaneous reproductive maturation. These results highlight the need to further investigate the role of ER on the reproductive process in prosobranch gastropods and whether this signalling pathway is prone to disruption by ECs.
    Aquatic Toxicology 11/2007; 84(4):465-8. · 3.73 Impact Factor
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