Ana D. Correia

Synergistic effects of the membrane actions of cecropin-melittin antimicrobial hybrid peptide BP100.

BP100 (KKLFKKILKYL-NH(2)) is a short cecropin A-melittin hybrid peptide, obtained through a combinatorial chemistry approach, which is highly effective in inhibiting both the in vitro and in vivo growth of economically important plant pathogenic Gram-negatives. The intrinsic Tyr fluorescence of BP100 was taken advantage of to study the peptides binding affinity and damaging effect on phospholipid bilayers modeling the bacterial and mammalian cytoplasmic membranes. In vitro cytotoxic effects of this peptide were also studied on mammalian fibroblast cells. Results show a stronger selectivity of BP100 toward anionic bacterial membrane models as indicated by the high obtained partition constants, one order of magnitude greater than for the neutral mammalian membrane models. For the anionic systems, membrane saturation was observed at high peptide/lipid ratios and found to be related with BP100-induced vesicle permeabilization, membrane electroneutrality, and vesicle aggregation. Occurrence of BP100 translocation was unequivocally detected at both high and low peptide/lipid ratios using a novel and extremely simple method. Moreover, cytotoxicity against mammalian models was reached at a concentration considerably higher than the minimum inhibitory concentration. Our findings unravel the relationships among the closely coupled processes of charge neutralization, permeabilization, and translocation in the mechanism of action of antimicrobial peptides.

Biochemical response in gilthead seabream (Sparus aurata) to in vivo exposure to a mix of selected PAHs

In our previous studies we assessed the biochemical response of juvenile seabream to the exposure to three selected polycyclic aromatic hydrocarbons (PAHs) (phenanthrene, pyrene and fluorene) in short-term laboratory experiments. Since in the analyses conducted in the field it is much more natural to expect the presence of multiple contaminants at the same time, the objective of this study was to analyse the reaction of seabream to the exposure to a mix of the three PAHs tested previously. The proportions of the individual components in the PAH mix used in our experiments were constant and determined on the basis of the concentration addition concept. We measured a set of hepatic biomarkers: 7-ethoxyresorufin-O-deethylase (EROD), glutathione-S-transferase (GST), catalase (CAT), superoxide dismutase (SOD) and lipid peroxidation; as well as biliary fluorescent aromatic compounds (FACs) metabolites. Moreover, we calculated the biotransformation index, the condition factor and the hepatosomatic index. Only a few of these indicators provided statistically significant response to the tested exposures, while FACs showed very strong increase with increasing concentration of the tested mix of PAHs. The results of this research are expected to contribute to the establishment of a good biochemical index of exposure to mixtures of PAHs in laboratory experiments, which can be further useful in field studies dealing with the impact of organic pollutants on marine organisms.

The joint effect of polycyclic aromatic hydrocarbons on fish behavior.

Polycyclic aromatic hydrocarbons (PAHs) are one of the most widespread organic environmental pollutants that pose a potential risk to marine biota. Although they occur as mixtures in the marine environment, only little information exists about their joint action on fish behavior. In 4-day tests with juvenile gilthead seabream (Sparus aurata) concentration-response analyses were performed for three PAH compounds--fluorene (FE), phenanthrene (PHE), and pyrene (PY). Responses of fish to these compounds were assessed by recording visually the changes in their locomotory activities and social behaviors. Based on these concentration-response data, mixture effects were predicted by applying the model of concentration addition. The mixture was tested using a fixed-ratio design, and the resulting effects were compared to the predictions. The single compounds and the mixture were accumulated in fish muscle and produced a clear change in the overall behavioral performance of fish, as all individual parameters were affected in a dose-response way. For lethargy and swimming, we determined regression fits for all single compounds, with PY the most potent (EC(10)=0.031 microM for swimming and 0.039 microM for lethargy) and FE the least (EC(10)=0.29 microM for swimming and 0.26 microM for lethargy). Also, changes in the number of lethargic fish were always the most sensitive parameter and social interaction the least. The mixture study revealed for lethargy and swimming a good agreement between the predicted and observed effect changes, and statistically significant deviations could not be identified.

Mixtures of estrogenic chemicals enhance vitellogenic response in sea bass.

Background The potential impact of natural and synthetic estrogens on aquatic ecosystems has attracted considerable attention because it is currently accepted that their joint effects are more severe when they are present in mixtures. Although it is well-known that they occur as mixtures in the marine environment, there is little information about the combined effects of estrogenic chemicals on marine biota. Objective In 14-day tests with juvenile sea bass, we analyzed singly and in combination the estrogenic activity of estradiol (E2), ethynylestradiol (EE2), and bisphenol A (BPA) using vitellogenin induction as an end point. Methods Fish were exposed to each compound, and on the basis of these concentration–response data, we predicted mixture effects by applying the model of concentration addition. The mixtures were tested using a fixed-ratio design, and the resulting mixture effects were compared to the predictions. Results EE2 was the most potent steroid, with an EC50 (median effective concentration) of 0.029 μg/L, 3.6 times more potent than E2 (EC50 = 0.104 μg/L); BPA was the least potent chemical, with an EC50 of 77.94 μg/L. The comparative assessment yielded a good agreement between observed and predicted mixture effects. Conclusions This study demonstrates the potential hazard of these compounds to seawater life by their ability to act together in an additive manner. It provides evidence that concentration addition can be used as a predictive tool for assessing the combined effects of estrogenic chemicals in marine ecosystems.

A Novel Behavioral Fish Model of Nociception for Testing Analgesics

Pain is a major symptom in many medical conditions, and often interferes significantly with a persons quality of life. Although a priority topic in medical research for many years, there are still few analgesic drugs approved for clinical use. One reason is the lack of appropriate animal models that faithfully represent relevant hallmarks associated with human pain. Here we propose zebrafish (Danio rerio) as a novel short-term behavioral model of nociception, and analyse its sensitivity and robustness. Firstly, we injected two different doses of acetic acid as the noxious stimulus. We studied individual locomotor responses of fish to a threshold level of nociception using two recording systems: a video tracking system and an electric biosensor (the MOBS system). We showed that an injection dose of 10% acetic acid resulted in a change in behavior that could be used to study nociception. Secondly, we validated our behavioral model by investigating the effect of the analgesic morphine. In time-course studies, first we looked at the dose-response relationship of morphine and then tested whether the effect of morphine could be modulated by naloxone, an opioid antagonist. Our results suggest that a change in behavioral responses of zebrafish to acetic acid is a reasonable model to test analgesics. The response scales with stimulus intensity, is attenuated by morphine, and the analgesic effect of morphine is blocked with naloxone. The change in behavior of zebrafish associated with the noxious stimulus can be monitored with an electric biosensor that measures changes in water impedance.

Antioxidant Capacity of the Essential Oils From Lavandula luisieri, L. stoechas subsp. lusitanica, L. stoechas subsp. lusitanica x L. luisieri and L. viridis Grown in Algarve (Portugal)

Abstract The antioxidant capacity of the essential oils isolated from the aerial parts of Lavandula luisieri, L. stoechas subsp. lusitanica, the hybrid L. stoechas subsp. lusitanica x L. luisieri, and L. viridis collected in different regions of Algarve (Portugal), as well as that of butylated hydroxytoluene (BHT), was evaluated using three different methods: free radical scavenging activity using 2,2-diphenyl-1-picrylhydrazyl (DPPH) method, thiobarbituric acid reactive substances assay (TBARS) and reductive potential. The chemical composition of the essential oils was analyzed by GC and GC/MS. The oils of L. luisieri were mainly constituted by 1,8-cineole (26–34%) and trans-α-necrodyl acetate (11–18%), only present in this lavender species. The oils of Lavandula stoechas subsp. lusitanica and the hybrid L. stoechas subsp. lusitanica x L. luisieri were dominated by fenchone (42–44%) and camphor (35–36%). Despite of the morphological resemblances of the L. stoechas subsp. lusitanica x L. luisieri with L. luisieri, no necrodane derivatives were detected in the hybrid, showing instead a major chemical similarity with L. stoechas oil. Lavandula viridis oil mainly comprised 1,8-cineole (33%) and camphor (20%). The oils of L. luisieri were the most effective as antioxidants, independent of the evaluation method used, although showing always lower antioxidant ability than BHT.