Application of a robust analytical method for quantifying progestins in environmental samples from three Portuguese Estuaries.

In the last years, progestins have raised special concerns for their documented negative effects on aquatic species, yet little is known about their environmental levels in surface waters and bioaccumulation in the trophic web. This study aimed to 1) adapt an extraction method for quantifying progestins in freeze-dried matrices, 2) validate the analytical procedure for three matrices: bivalve, polychaete, and crustacean, and 3) characterize levels of the four most prescribed synthetic progestins in key species across three Portuguese estuaries. Through the validated method, progestins were only quantifiable for the crustacean. Values were generally low, peaking with drospirenone values in Ria de Aveiro (1.33 ± 0.26 ng/g ww) and Tagus estuary (1.42 ± 0.55 ng/g ww), while Ria Formosa exhibited the lowest progestin concentrations (< 1 ng/g ww). This study enabled the development of a precise extraction and analytical method for quantifying steroid hormones in three distinct biological matrices.

Baseline progestins characterization in surface waters of three main Portuguese estuaries.

Synthetic progestins are micropollutants of special concern, due to their growing use in human and veterinary therapies and their risks to aquatic life. Currently, there is a lack of environmental information on these compounds, worldwide. The main objective of this work was to characterize the levels of the most consumed progestins in Portugal. For that, Ria de Aveiro, Tagus estuary and Ria Formosa were sampled in a temporal perspective to evaluate levels of drospirenone (DRO), desogestrel (DSG), gestodene (GST) and levonorgestrel (LNG). Drospirenone and desogestrel were the most abundant progestins. In the North of Portugal, DSG was the most abundant (Aveiro: 193.9 ng L-1 in summer), while DRO was more representative in the South (Tagus: 178.9 ng L-1; Formosa: 125.7 ng L-1) and also in summer. These spatial differences can be associated with the hydrodynamics of each estuarine system as well as the distinct population and tourist levels associated with each site.

Combined effects of climate change and environmentally relevant mixtures of endocrine disrupting compounds on the fitness and gonads' maturation dynamics of Nucella lapillus (Gastropoda).

Coastal areas are affected by multiple stressors like climate change and endocrine disruptors (EDCs). In the laboratory, we investigated the combined effects of increased temperature and EDCs (drospirenone and mercury) on the fitness and gonads' maturation dynamics of the marine gastropod Nucella lapillus for 21 days. Survival was negatively affected by all the stressors alone, while, in combination, a synergistic negative effect was observed. Both chemicals, as single factors, did not cause any effect on the maturation stage of ovaries and testis. However, in the presence of a higher temperature, it was clear a delay in the maturation stage of the ovaries, but not in the testis, suggesting a higher negative impact of the stressors in females than in males. In summary, drospirenone caused a low negative impact in aquatic species, like gastropods, but in combination with other EDCs and/or increased temperature can be a matter of concern.

Genotoxic effects of combined multiple stressors on Gammarus locusta haemocytes: Interactions between temperature, pCO2 and the synthetic progestin levonorgestrel.

Climate change and pharmaceutical contamination are two priority research topics due to their impacts in the aquatic ecosystems and in the food chain structure. In the bottom of many food chains are the invertebrates, like the amphipods, which are important environmental and ecotoxicological models. In this study, we combined the increase of temperature [ambient and warming temperature], pCO2 [normocapnia and hypercapnia] and the synthetic progestin levonorgestrel (LNG) [environmentally relevant concentration (10 ng L-1) and 100-fold higher (1000 ng L-1)] to evaluate the genotoxic effects on the amphipod Gammarus locusta haemocytes, using the comet assay technique. Additionally, the study examined protective/potentiating effects of the three tested factors against hydrogen peroxide (H2O2)-induced DNA damage in haemocytes after ex vivo exposure. Our data revealed no significant effects of any of the three stressors on DNA damage of G. locusta haemocytes or protection against H2O2-induced DNA damage after twenty-one days exposure. Only a significant effect of the solvent was visible, since it was able to induce higher DNA damage (i.e. strand breaks) on exposed individuals. On the other hand, LNG exposure seemed to induce a slight increase of DNA damage after H2O2 exposure. Our findings suggest that more short-term studies to conclude about the genotoxicity and/or protective effects of the stress factors in G. locusta should be made, attending to the fast turnover rate of repairing cells that could have masked impacts seen only after the end of the experiment.

Cytological, immunocytochemical, ultrastructural and growth characterization of the rainbow trout liver cell line RTL-W1.

Despite its wide use in toxicology, a detailed characterization of RTL-W1 cell line lagged behind leaving ambiguities about its cell origin. We aimed to better characterize the line regarding cell phenotype and tumorigenic state. We studied RTL-W1 cells in monolayers and in (4-22-week-old) aggregates considering: (a) morphology (light and electron microscopy); (b) immunophenotype using AE1/AE3, vimentin, Cam5.2, CK7 and CK19 and e-cadherin antibodies and (c) growth behavior. RTL-W1 organelle content is constituted basically by mitochondria and abundant free ribosomes, with no (cytochemically) detectable peroxisomes and lysosomes. Immunocytochemistry showed a strong marking for AE1/AE3 and vimentin (in a cell subset). Since AE1/AE3 stained biliary epithelial ducts in trout liver, and considering the morphological characteristics and long term culture, RTL-W1 cells seem more similar to bile preductular epithelial cells (considered as stem cells in teleost liver). Also, we observed abnormal nuclear features described for both malignant cell lines and stem cells, so we could not conclude about tumorigenicity. Cell aggregates had signs of hepatocytic differentiation, such as the development of RER and microvillus-like projections into intercellular spaces. The morphological resemblance to the original tissue suggests that aggregates could have an added value in metabolic as well as in cell-to-cell interaction studies.

Effects of ethinylestradiol and of an environmentally relevant mixture of xenoestrogens on steroidogenic gene expression and specific transcription factors in zebrafish.

In natural environments fish are exposed to endocrine disrupting compounds (EDCs) present at low concentrations and with different modes of actions. Here, adult zebrafish of both sexes were exposed for 21 days to an estrogenic mixture (Mix) of eleven EDCs previously quantified in Douro River estuary (Portugal) and to 100 ng/L 17α-ethinylestradiol (EE2) as positive control. Vitellogenin mRNA and HSI in males confirmed both exposure regimes as physiologically active. Potential candidates for estrogenic disturbance of steroidogenesis were identified (StAR, 17ß-HSD1, cyp19a1), but Mix only affected cyp19a1 in females. Significant differences in the response of FSHß, cypa19a2, 20ß-HSD were observed between EE2 and Mix. Mtf-1 and tfap2c transcription factor binding sites were discovered in the putative promoter regions and corresponding transcription factors were found to be differentially expressed in response to Mix and EE2. The results suggest that "non-classical effects" of estrogenic EDC in fish are mediated via transcription factors.

[Contributions to the history of burn treatment]. / Contributos para a história do tratamento de queimados.

The authors make a brief review of burn treatment in Portugal as well as describing the Burn Unit of the Coimbra University Hospitals. The scientific advances in this area and new perspectives to the future are also mentioned, emphasizing the importance of burn prevention and a complete social reintegration of the burn patient.

[Avulsion of the skin of the penis and scrotum]. / Avulsão cutânea péno-escrotal.

The authors present a case report of traumatic avulsion of the skin of the penis and scrotum. Surgical treatment was performed in one surgical stage a few hours after the accident. The skin repair of the penis was carried out with a skin graft, while scrotum reconstruction was performed with fasciocutaneous flaps of the inner face of the thighs, with supero-medial pedicle, achieving a good result. The authors justify their choice of procedure, also mentioning the other methods most employed.

[Microsurgical reconstruction of the head and neck: experience of 63 free flaps]. / Reconstrução microcirúrgica da cabeça e pescoço.

The authors present the experience of 63 clinical cases of microsurgical reconstruction of the head and neck. These reconstructions were accomplished by different microvascular free flaps which were selected according to the type, size and location of the defects. Fasciocutaneous, osteofasciocutaneous, adipofascial, myo, myocutaneous, myosseous, jejunum and great omentum flaps were applied. The etiology was traumatic in 10 cases, tumoral in 41 cases, congenital in 7 cases, iatrogenic in 3 cases and infectious in 2 cases. The reconstructed anatomical regions were the scalp in 6 cases, the skull base in 1 case, the orbit in 3 cases, the nose in 1 case, the hemiface in 7 cases, the lips in 1 case, the intraoral cavity in 21 cases, the mandible in 8 cases, the intraoral cavity plus mandible in 11 cases and the cervical esophagus in 1 cases. An analysis was made of surgical times, selection and survival rate of the free flaps and the morphofunctional quality of the reconstructions. Considering the good quality of reconstructions, the amelioration of quality of life and the low morbidity in these patients, microvascular free flaps are considered a first choice technique for head and neck reconstruction when the size and location of the defect prevents the use of local flaps.