Estudo do padrão de esteróides em "Mytilus" sp. : Relação com o ciclo reprodutivo e com a regulação hidromineral

Dissertação de Doutoramento em Ciências Biomédicas, área de especialização em Fisiologia, apresentada ao Instituto de Ciências Biomédicas de Abel Salazar da Universidade do Port

Ciclo anual de oogénesis en Lipophrys pholis (Pisces: Blenniidae)

Lipophrys pholis has been shown to be responsive to a variety of environmental contaminants, some of them able to impair reproduction. Description of the normal cycle of oogenesis of this newly proposed sentinel species is important since this data may function as a baseline for comparison in ecotoxicological studies, among other applications. Based on histological observations, L. pholis ovarian development in adult is asynchronous, and 7 ovarian germ cells can be described (oogonia, early and late perinuclear oocytes, cortical-alveolar oocytes, early vitellogenic oocytes, vitellogenic oocytes and spawning oocytes). Using a stereological approach together with the morphologic characteristics of ovarian cells, the ovarian cycle of L. pholis was divided into 3 maturation stages: early oogenesis (May); mid-oogenesis (September), and spawning (November to January). Ovarian cell proportions and gonadosomatic index confirmed that the reproductive period of L. pholis near the southern limit of distribution of the species occurs during cold-water periods, between November and May. The collected data will help to fill some of the gaps in information that still exist on L. pholis oogenesis, thus allowing a better integration of this species as a sentinel for the detection of contaminants in European coastal waters.En trabajos recientes, se ha visto que Lipophrys polis es sensible a diversos contaminantes medioambientales, algunos de los cuales pueden afectar a la reproducción. La descripción del ciclo normal de oogénesis de esta nueva propuesta de especie centinela es importante, ya que serviría como referencia para futuros estudios toxicológicos, entre otras aplicaciones. En base a observaciones histológicas, se pueden describir siete estados de los oocitos (oogonia, estado perinuclear temprano y tardío, estado cortico-alveolar, vitelogénesis temprana, vitelogénesis y oocitos desovados). Desde un enfoque estereológico, junto con las características morfológicas de las células ováricas, el ciclo ovárico de L. polis se ha dividido en tres estados de maduración: oogénesis temprana (mayo), oogénesis media (septiembre) y desove (de noviembre a enero). Las proporciones de las células ováricas y el índice gonadosomático, confirmaron que el periodo reproductivo de L. polis, en el límite meridional de distribución de la especie, se produce durante los periodos de agua fría, entre noviembre y mayo. Los datos obtenidos ayudarán a cubrir algunas de las lagunas que todavía existen en el conocimiento de la ovogénesis de L. pholis, permitiendo, por tanto, una mejor integración de esta especie como centinela para la detección de contaminantes en las aguas costeras europeas

Seasonal patterns of polycyclic aromatic hydrocarbons in digestive gland and arm of octopus (Octopus vulgaris) from the Northwest Atlantic

Among organic pollutants existing in coastal areas, polycyclic aromatic hydrocarbons (PAHs) are of great concern due to their ubiquity and carcinogenic potential. The aim of this study was to evaluate the seasonal patterns of PAHs in the digestive gland and arm of the common octopus (Octopus vulgaris) from the Northwest Atlantic Portuguese coast. In the different seasons, 18 PAHs were determined and the detoxification capacity of the species was evaluated. Ethoxyresorufin O-deethylase (EROD) and ethoxycoumarin O-deethylase (ECOD) activities were measured to assess phase I biotransformation capacity. Individual PAH ratios were used for major source (pyrolytic/petrogenic) analysis. Risks for human consumption were determined by the total toxicity equivalence approach. Generally, low levels of PAHs were detected in the digestive gland and in the arm of octopus, with a predominance of low molecular over high molecular weight compounds. PAHs exhibited seasonality in the concentrations detected and in their main emission sources. In the digestive gland, the highest total PAH levels were observed in autumn possibly related to fat availability in the ecosystem and food intake. The lack of PAH elimination observed in the digestive gland after captivity could be possibly associated to a low biotransformation capacity, consistent with the negligible/undetected levels of EROD and ECOD activity in the different seasons. The emission sources of PAHs found in the digestive gland varied from a petrogenic profile observed in winter to a pyrolytic pattern in spring. In the arm, the highest PAH contents were observed in June; nevertheless, levels were always below the regulatory limits established for food consumption. The carcinogenic potential calculated for all the sampling periods in the arm were markedly lower than the ones found in various aquatic species from different marine environments. The results presented in this study give relevant baseline data for environmental monitoring of organic pollution in coastal areas

Toxicity of seven priority hazardous and noxious substances (HNSs) to marine organisms: Current status, knowledge gaps and recommendations for future research

Shipping industry and seaborne trade have rapidly increased over the last fifty years, mainly due to the continuous increasing demand for chemicals and fuels. Consequently, despite current regulations, the occurrence of accidental spills poses an important risk. Hazardous and noxious substances (HNSs) have been raising major concern among environmental managers and scientific community for their heterogeneity, hazardous potential towards aquatic organisms and associated social-economic impacts. A literature review on ecotoxicological hazards to aquatic organisms was conducted for seven HNSs: acrylonitrile, n-butyl acrylate, cyclohexylbenzene, hexane, isononanol, trichloroethylene and xylene. Information on the mechanisms of action of the selected HNS was also reviewed. The main purpose was to identify: i) knowledge gaps in need of being addressed in future research; and ii) a set of possible biomarkers suitable for ecotoxicological assessment and monitoring in both estuarine and marine systems. Main gaps found concern the scarcity of information available on ecotoxicological effects of HNS towards marine species and their poorly understood mode of action in wildlife. Differences were found between the sensitivity of freshwater and seawater organisms, so endpoints produced in the former may not be straightforwardly employed in evaluations for the marine environment. The relationship between sub-individual effects and higher level detrimental alterations (e.g. behavioural, morphological, reproductive effects and mortality) are not fully understood. In this context, a set of biomarkers associated to neurotoxicity, detoxification and anti-oxidant defences is suggested as potential indicators of toxic exposure/effects of HNS in marine organisms. Overall, to support the development of contingency plans and the establishment of environmental safety thresholds, it will be necessary to undertake targeted research on HNS ecotoxicity in the marine environment. Research should address these issues under more realistic exposure scenarios reflecting the prevailing spatial and temporal variability in ecological and environmental conditions

Retinol Metabolism in the Mollusk Osilinus lineatus Indicates an Ancient Origin for Retinyl Ester Storage Capacity

Although retinoids have been reported to be present and active in vertebrates and invertebrates, the presence of mechanisms for retinoid storage in the form of retinyl esters, a key feature to maintain whole-organism retinoid homeostasis, have been considered to date a vertebrate innovation. Here we demonstrate for the first time the presence of retinol and retinyl esters in an invertebrate lophotrochozoan species, the gastropod mollusk Osilinus lineatus. Furthermore, through a pharmacological approach consisting of intramuscular injections of different retinoid precursors, we also demonstrate that the retinol esterification pathway is active in vivo in this species. Interestingly, retinol and retinyl esters were only detected in males, suggesting a gender-specific role for these compounds in the testis. Females, although lacking detectable levels of retinol or retinyl esters, also have the biochemical capacity to esterify retinol, but at a lower rate than males. The occurrence of retinyl ester storage capacity, together with the presence in males and females of active retinoids, i.e., retinoic acid isomers, indicates that O. lineatus has a well developed retinoid system. Hence, the present data strongly suggest that the capacity to maintain retinoid homeostasis has arisen earlier in Bilateria evolution than previously thought

ABC transporters in fish species: a review

ATP-binding cassette (ABC) proteins were first recognized for their role in multidrug resistance (MDR) in chemotherapeutic treatments, which is a major impediment for the successful treatment of many forms of malignant tumors in humans. These proteins, highly conserved throughout vertebrate species, were later related to cellular detoxification and accounted as responsible for protecting aquatic organisms from xenobiotic insults in the so-called multixenobiotic resistance mechanism (MXR). In recent years, research on these proteins in aquatic species has highlighted their importance in the detoxification mechanisms in fish thus it is necessary to continue these studies. Several transporters have been pointed out as relevant in the ecotoxicological context associated to the transport of xenobiotics, such as P-glycoproteins (Pgps), multidrug-resistance-associated proteins (MRPs 1-5) and breast cancer resistance associated protein (BCRP). In mammals, several nuclear receptors have been identified as mediators of phase I and II metabolizing enzymes and ABC transporters. In aquatic species, knowledge on co-regulation of the detoxification mechanism is scarce and needs to be addressed. The interaction of emergent contaminants that can act as chemosensitizers, with ABC transporters in aquatic organisms can compromise detoxification processes and have population effects and should be studied in more detail. This review intends to summarize the recent advances in research on MXR mechanisms in fish species, focusing in (1) regulation and functioning of ABC proteins; (2) cooperation with phase I and II biotransformation enzymes; and (3) ecotoxicological relevance and information on emergent pollutants with ability to modulate ABC transporters expression and activity. Several lines of evidence are clearly suggesting the important role of these transporters in detoxification mechanisms and must be further investigated in fish to underlay the mechanism to consider their use as biomarkers in environmental monitoring

Triphenyltin and tributyltin, single and in combination, promote imposex in the gastropod Bolinus brandaris

8 pages, 2 tablesSpecimens of Bolinus brandaris (neogastropod) were injected with a single dose of 500 ng/g body weight (b.w.) of tributyltin chloride (TBTCl) or triphenyltin chloride (TPTCl), or a mixture of both compounds (250 ng TBT/g b.w.+250 ng TPT/g b.w.), for a period of up to 31 days. At the end of 4 weeks, significant increases in the female penis size of those gastropods injected with TBT (Po0:05), TPT (Po0:05), or the mixture TBT+TPT (Po0:01) were recorded. In parallel, a group of animals was injected with the neuropeptide APGWamide but this compound failed to promote imposex, suggesting that APGWamide is not involved in imposex promotion in B. brandaris. Acetylcholinesterase activity, a biomarker of neurotoxicity, was determined in the neuroganglia at the end of the experiment, but no significant differences among treatments were found. Overall, these results support the hypothesis that TPT also acts as an endocrine disrupter in this neogastropod species. Our observations also highlight, for the first time, synergistic effects of organotin mixtures having imposex promotion as an endpoint.This work was partly possible through a visit grant to M.M. Santos from ‘‘Junta de Andalucía.’’ M.M. Santos also acknowledged financial support from the project ‘‘Portuguese Foundation for Science and Technology PDCTM/MAR/15284/99.’’Peer reviewe

Histopathological lesions, P-glycoprotein and PSNA expression in zebrafish (Danio rerio) liver after a single exposure to diethylnitrosamine

The presence of carcinogenic compounds in the aquatic environment is a recognized problem. ABC transporters are well known players in the multidrug-resistance (MDR) phenomenon in mammals associated with resistance to chemotherapy, however little is known in fish species. Thus, the aim of this study was to induce hepatic tumours and evaluate long-term effects on P-glycoprotein (P-gp) and proliferating cell nuclear antigen (PCNA) proteins in Danio rerio liver, after exposure to diethylnitrosamine (DEN). Several hepatic histopathological alterations were observed in zebrafish after exposure to DEN including pre-neoplastic lesions 6 and 9 months post-exposure. After 3, 6 and 9 months of exposure to DEN, P-gp and PCNA proteins expression were up-regulated. In conclusion, this study has shown that zebrafish ABC transporters can play a similar role as in human disease, hence zebrafish can be used also as a biological model to investigate in more deep mechanisms involved in disease processes

Stress biomarkers in juvenile Senegal Sole, Solea senegalensis, exposed to the water-accommodated fraction of the "Prestige" fuel oil

5 pages, 1 table.-- PMID: 17924040 [PubMed].-- Available online Oct 9, 2007.The "Prestige" oil spill occurred in November 2002 off the Galician Coast (NW Spain). Soon after the black tide episode, that caused an ecological disaster, several monitoring programs and projects evaluating the acute and longterm toxicity of the crude oil were initiated (see monograph Marine Pollution Bulletin 2006; vol. 53). Characteristics of the "Prestige" oil are described in Albaigés and Bayona (2003). Briefly, it consists of 50% polycyclic aromatic hydrocarbons (PAHs), mostly dominated by 2–3 ringed compounds which, contrarily in contrast to the higher molecular weight PAHs, are not reported as very toxic to fish (Seruto et al. 2005). Also, due to the heavy nature of this oil and its low solubility in water, the dispersion in sea water was very low and it mostly remained in situ forming oil patches adhered to rocks and sediment.Fish, as with all aerobic aquatic organisms, face natural stress conditions when oxygen is not fully reduced, and intermediate reactive oxygen species (ROS) are formed. Enhanced ROS formation can also take place under exposure to some toxicants such as crude oil (Livingstone 2001). In particular, the water-accommodated fraction (WAF) of some crude oils has been responsible for oxidative stress situations in aquatic organisms (Cajaraville et al. 1992). Hence, in order to evaluate if exposure to the WAF of the "Prestige" crude would interfere with the antioxidant defense system of juvenile sole, Solea senegalensis, the activities of indicative enzymes were analyzed. These included: catalase (CAT), total glutathione peroxidase (t-GPX; sum of Selenium dependent and independent forms), glutathione reductase (GR) and DT-diaphorase (DTD), as part of the antioxidant defense system. However, when this natural protective system is saturated, damage to key molecules such as DNA, proteins or lipids can occur. The latter, termed lipid peroxidation (LP), was included in the present study as a biomarker of effect, indicating damage caused by ROS. The enzyme acetylcholinesterase (AChE) regulates nerve impulse transmissions and is used as a marker of exposure to neurotoxic substances (Galloway et al. 2006). Carboxylesterases (CbE) constitute an heterogeneous group of isozymes, dominant in the liver, that play a role in the metabolism and subsequent detoxification of many xenobiotics. In marine fish AChE and CbE are biomarkers used as indicators of neurotoxicity (Lionetto et al. 2004) and enhanced hepatic metabolism (Wheelock et al. 2005). The phase II conjugating enzyme glutathione S-transferase (GST) has also been included in the present work as a measure of the detoxification metabolism.This study is part of a project aiming to evaluate the toxicity of the water-accommodated fraction (WAF) of the "Prestige" oil in juvenile Solea senegalensis after short-term (24, 48 and 72 h) exposure to environmentally realistic PAHs levels. The choice of this species was based on (1) its high economic interest in the affected region, (2) its benthic nature and, therefore, its adequacy for pollution monitoring programs and (3) its ease to rear under laboratory conditions.The present study was funded by the Spanish project VEM2003-20068-CO5-01 and a CSIC-GRICES agreement.Peer reviewe

Esterases activities and lipid peroxidation levels in muscle tissue of the shanny Lipophrys pholis along several sites from the Portuguese Coast

9 pages, 2 figures, 4 tables.-- PMID: 18295805 [PubMed].-- Printed version published May 2008.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 μM), 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.The present study was funded by a CSIC-GRICES agreement and Interreg III B “Atlantic Area” project EROCIPS.Peer reviewe