Maria Armanda Reis-Henriques

Polycyclic aromatic hydrocarbons inhibit in vitro ovarian steroidogenesis in the flounder (Platichthys flesus L.)

The in vitro effect of polycyclic aromatic hydrocarbons (PAHs) on ovarian steroidogenesis of the flounder (Platichthys flesus L.) was determined. Fully vitellogenic ovary tissue was in vitro incubated in the presence of phenanthrene, benzo[a]pyrene or chrysene, using 17alpha-hydroxyprogesterone or androstenedione as precursors. Androstenedione (A), testosterone (T) and 17beta-estradiol (E(2)) synthesised in the presence of PAHs were assayed by radioimmunoassay and results compared with control incubations. In order to establish the effect of PAHs on the steroidogenic enzyme systems cytochrome P450 17,20-lyase (P450-17,20l), 17beta-hydroxysteroid dehydrogenase (17beta-HSD) and cytochrome P450 aromatase (P450-arom), results were also compared with the action of ketoconazole (KCZ) and aminoglutethimide (AMG), wich are, respectively, inhibitors of cytochrome P450 steroidogenic enzymes and of P450-arom. KCZ inhibited secretion of A and E(2) in 65% and T in 40%, as a consequence of inhibited P450-17,20l and P450-arom. AMG inhibited P450-arom, which resulted in decreased E(2) synthesis to approximately 50% of control incubations. All the three PAHs inhibited A secretion by approximately 50% and E(2) from 10 to 40%. Because steroid conjugation was also inhibited by phenanthrene, it could be concluded that PAH action was mediated by an inhibitory effect over P450-17,20l, 17beta-HSD and P450-arom. Except for 17beta-HSD, PAHs resembled KCZ, and P450-17,20l was the most sensitive to their inhibitory effect. In conclusion, PAHs strongly blocked the activity of P450-17,20l, a rate-limiting enzyme for conversion of C21 to C19 steroids, and showed, therefore, the potential to disrupt the reproductive cycle of fish living in polluted environments, due to impairment of steroid biosynthesis.

Replacement of fish meal by plant proteins in the diet of Nile tilapia: digestibility and growth performance

Apparent digestibility coefficient (ADC) values for a number of ingredients of plant or animal origin were obtained in order to formulate diets based on such values and to evaluate growth performance of Nile tilapia Oreochromis niloticus fed four experimental diets in which fish meal was gradually replaced by a mixture of other ingredients.The digestibility of various diet components was measured by using an inert marker in the feed and by using the Guelph faeces collecting system. ADC values of the ingredients tested were generally high, especially for fish meal. It was found that extruded pea seed meal (92.6%), defatted soybean meal (94.4%), full-fat toasted soybean (90.0%) and micronized wheat (88.6%) were the best vegetable proteins tested. Lupin seed meal and faba bean meal had similar ADC values for protein and energy.Groups of tilapia, initial mean body weight (SD) 6.7 (0.1) g, were fed experimental diets with the same digestible protein (DP) and digestible energy (DE) containing graded levels of a mixture of vegetable ingredients as partial or total replacement of fish meal protein. A growth trial was conducted over 12 weeks as partial or total replacement of fish meal protein. A growth trial was conducted over 12 weeks at a water temperature of 25 °C. Significant differences were observed for weight gain among tilapia fed diets D0, D33, D66 and D100 (containing only animal protein, 33, 66, and 100% of plant protein, respectively). No significant differences were observed for voluntary intake among tilapia fed diets D0, D33 and D66. These values were significantly lower than those observed for tilapia fed plant protein based diet (D100) and suggest the possibility of partial replacement of fish meal by vegetable proteins without negative effects.

Studies of endogenous steroids from the marine mollusc Mytilus edulis L. By gas chromatography and mass spectrometry

1. 1. Identification of endogeneous steroids in Mytilus edulis (progesterone, androstenedione, testosterone, 5α-dihydrotestosterone, estradiol-17β, estrone, androsterone and androstanediol) in male and female specimens, was carried out by gas chromatography-mass spectrometry. 2. 2. The levels of progesterone, androstenidione, testosterone, estradiol-17β and estrone were estimated by means of RIA in specimens collected at two different months of the year. 3. 3. The levels of progesterone and androstenedione are higher than those of the other steroids, and show a different pattern between both samples. 4. 4. These differences allow us to suggest a possible function of these steroids in the reproduction of Mytilus edulis.

Plasma steroid levels in female flounder (Platichthys flesus) after chronic dietary exposure to single polycyclic aromatic hydrocarbons

The chronic effects of polycyclic aromatic hydrocarbons (PAHs) on ovary development, total hepatic lipids and plasma sex- and corticosteroid levels in female flounder (Platichthys flesus) were examined. Sexually mature feral female flounder were exposed via the diet to phenanthrene (0.5, 2.5 or 12.5 nmol/g food) or chrysene (0.4 nmol/g food) for 12 weeks, during the previtellogenic phase of the annual reproductive cycle. PAH exposure did not directly affect germ cell development since no structural and/or developmental differences were observed between control and exposed fish. On the contrary, all treatments resulted in altered plasma steroid levels. The most pronounced effect was the significant decrease in plasma 17 beta-estradiol to 19 +/- 11%, 27 +/- 7%, 63 +/- 20% and 61 +/- 12% in relation to control fish, respectively, in flounders exposed to 12.5, 2.5 or 0.5 nmol phenanthrene/g food and 0.4 nmol chrysene/g food. Impaired ovarian growth was not observed, most likely because experiments were ended before the period of vitellogenesis, even though a non-significant general decline in total hepatic lipids could be observed. Moreover, all exposed flounders, except fish fed with the highest amount of phenanthrene, showed a negative correlation between plasma 17 beta-estradiol and 17 alpha-hydroxyprogesterone levels (r = -0.46). One possible explanation is that PAH action may be mediated by a specific inhibition of steroidogenic enzymes. These findings provide evidence that selected PAHs are antiestrogenic xenobiotics with the capability to impair female teleost reproductive function.

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.

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.

Assessment of contaminants and biomarkers of exposure in wild and farmed seabass

This study aimed to investigate the relationships between levels of organochlorine compounds (PCBs and DDTs), polycyclic aromatic hydrocarbons (PAHs), metals and biomarkers of exposure (EROD and GST) and oxidative stress biomarkers, in wild and cultured European seabass (Dicentrarchus labrax). Additionally, the fatty acid profile was determined in muscle to assess differences between cultivated and wild specimens of marketable size. Although results pointed to relatively low concentration of contaminants, wild specimens presented enhanced accumulation of metals and cultivated specimens higher levels of organic compounds. Biomarkers were correlated with contaminants in tissues, showing that higher levels can cause effects at the biochemical level. Wild specimens have presented higher content on omega-3 fatty acids in muscle. Besides the useful application of biomarkers in wild species, this study has shown that they can also be applied to monitor biological effects in the fish farming process.

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.

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.

Metal accumulation and oxidative stress responses in, cultured and wild, white seabream from Northwest Atlantic

Metals are environmentally ubiquitous and can be found at high concentrations in seawater and subsequently in marine organisms. Metals with high redox potential can trigger oxidative stress mechanisms with damaging effects in biological tissues. In aquatic species, oxidative stress has been evaluated by assessing antioxidant enzymes activities and oxidative damages in tissues. The purpose of this study was to evaluate oxidative stress biomarkers and metal residues in white seabream (Diplodus sargus), a species entering aquaculture production in Portugal. Metal residues (Cu, Cd, As and Pb), in liver and muscle, as well as oxidative stress biomarkers were assessed at different stages in the life cycle of white seabream under culture conditions and in wild specimens, of a marketable size. Metal concentrations in tissues were low, and below the established limits. However, wild white seabream showed higher accumulation than cultured ones. Antioxidant enzymes, namely catalase (CAT) and superoxide dismutase (SOD), were correlated with metal accumulation. Oxidative damages to tissues were low, with wild white seabream showing lower levels than cultured fish. This study showed that white seabream has a good antioxidant defense system, capable of reducing oxidative damages in tissues resulting from the presence of metals.