Avaliação do potencial cancerigénico de microcistinas (cianotoxinas)

Tese de doutoramento em Farmácia (Toxicologia), apresentada à Faculdade de Farmácia da Universidade de Lisboa, 2009.

Occupational exposure to mycotoxins: aspects to consider for the aggregate and cumulative risk assessment

Mycotoxins are an important group of naturally occurring substances known to contaminate a huge variety of agricultural products, feed and food commodities. The main concern is their widespread presence and toxic effects on humans and animals as they have been described as cytotoxic, nephrotoxic, hepatotoxic, teratogenic, immunosuppressive, mutagenic and/or carcinogenic. However, until now, risk assessments and regulations have usually been performed on individual mycotoxins despite humans and animals are being frequently exposed to a multitude of mycotoxins simultaneously. Moreover, even though some exposures through inhalation and dermal contact may potentially occur, only oral ingestion has been considered as the sole route of exposure in all the evaluations. However, more recent studies have also demonstrated airborne exposure to mycotoxins in different occupational settings with emphasis on agricultural professions. In these cases, skin contact with mold-infested substrates and inhalation of spore-borne toxins are the most important sources of exposure. Still, mycotoxins are not normally recongnize as na occupational hazard and exposure is different from the one ocurring by food intake. In this case, exposure is charaterized to be acute and simultaneous to other mycotoxins and also to fungi and dust. All these features increase the challenge implicated in the risk assessment process. Some topics will be presented and discussed in detailed such as: What occupational settings should be consider in this case; possible exposure routes; exposure characterization; how to assess exposure; co-exposure; aggregate exposure and cumulative risk assessment.

Twenty years of Hendra virus: laboratory submission trends and risk factors for infection in horses

Hendra virus (HeV) was first described in 1994 in an outbreak of acute and highly lethal disease in horses and humans in Australia. Equine cases continue to be diagnosed periodically, yet the predisposing factors for infection remain unclear. We undertook an analysis of equine submissions tested for HeV by the Queensland government veterinary reference laboratory over a 20-year period to identify and investigate any patterns. We found a marked increase in testing from July 2008, primarily reflecting a broadening of the HeV clinical case definition. Peaks in submissions for testing, and visitations to the Government HeV website, were associated with reported equine incidents. Significantly differing between-year HeV detection rates in north and south Queensland suggest a fundamental difference in risk exposure between the two regions. The statistical association between HeV detection and stockhorse type may suggest that husbandry is a more important risk determinant than breed per se. The detection of HeV in horses with neither neurological nor respiratory signs poses a risk management challenge for attending veterinarians and laboratory staff, reinforcing animal health authority recommendations that appropriate risk management strategies be employed for all sick horses, and by anyone handling sick horses or associated biological samples.

The effects of PVP(FE(III)) catalyst and polymer molecular weight on gene delivery via biodegradable crosslinked polyethylenimine

Human gene therapy has faced many setbacks due to the immunogenicity and oncogenity of viruses. Safe and efficient alternative gene delivery vehicles are needed to implement gene therapy in clinical practice. Polymeric vectors are an attractive option due to their availability, simple chemistry, and low toxicity and immunogenicity. Our group has previously reported biodegradable polyethylenimines (PEI) that show high transfection efficiency and low toxicity by cross-linking 800 Da PEI with diacrylate cross-linkers using Michael addition. However, the synthesis was difficult to control, inconsistent, and resulted in polymers with a narrow range of molecular weights. In the present work, we utilized a heterogenous PVP(Fe(III)) catalyst to provide a more controllable PEI crosslinking reaction and wider range of biodegradable PEIs. The biodegradable PEIs reported here have molecular weights ranging from 1.2 kDa to 48 kDa, are nontoxic in MDA-MB-231 cells, and show low toxicity in HeLa cells. At their respective optimal polymer:DNA ratios, these biodegradable PEIs demonstrated about 2-5-fold higher transfection efficiency and 2-7-fold higher cellular uptake, compared unmodified 25 kDa PEI. The biodegradable PEIs show similar DNA condensation properties as unmodified PEI but more readily unpackage DNA, based on ethidium bromide exclusion and heparan sulfate competitive displacement assays, which could contribute to their improved transfection efficiency. Overall, the synthesis reported here provides a more robust, controlled reaction to produce cross-linked biodegradable PEIs that show enhanced gene delivery, low toxicity, and high cellular uptake and can potentially be used for future in vivo studies.

Avaliação da ecotoxicidade de percolados em áreas de disposição de resíduos na região metropolitana de Natal/RN

Leachates are effluent produced by decomposition of solid waste, they have complex composition and can be highly toxic. Therefore such percolated liquid should be collected and treated properly to avoid environmental contamination of soil and of water bodies. The objective of this study was to evaluate the toxicity through ecotoxicological tests with Ceriodaphnia dubia (Cladocera - Crustacea) of percolated liquids generated in two different systems of municipal solid waste (MSW) disposal in the city of Natal/ RN: A Sanitary Landfill in the Metropolitan Region of Natal/ RN, and in a dump off area. Furthermore, it was evaluated the possible contamination of the underground water of the dump off area. Two monthly samples were taken at four points between the months of May/2009 and January/2010. The Point "A" corresponds to the end of the pond leachate treatment in ASRMN; The Point "B" corresponds to a containment pond at the dump. The Point "C" is an area near one of the cells of the dump off area where the leachate outcrops; The Point "D" stands for an underground water well at the area. The last point, called "E" was sampled only once and corresponds to the slurry produced by temporary accumulation of solid waste in the open area of the dump. The ecotoxicological tests, acute and chronic, followed the ABNT 13373/2005 rules, with some modifications. The samples were characterized by measuring the pH number, the dissolved oxygen (DO), the salinity, BOD5, COD, Cd, Cu, Pb, Cr, Fe, Mg, Ni, and Zn. At Point A, the average number of EC50-48h ranged between 1.0% and 2.77% (v/v), showing a high toxicity of the leachate to C.dubia in all months. To this point, positive correlations were found between the EC50- 48 with precipitation. Negative correlations were found between the EC50- 48h with salinity. At point B there was no response of the acute exposure of organisms to the test samples. At point C the EC50-48h ranged from 17.68% to 35.36% in just two months of the five ones analyzed, not correlated meaning. Point D, the EC50-48h level ranged between 12.31% and 71.27%, showed a negative correlation with, only, precipitation. Although it was observed toxicity of underground water in the Landfill Area, there was no evidence of water contamination by leachate, however, due to the toxic character of this water, additional tests should be conducted to confirm the quality of water that is used for human supply. At point E there was no acute toxicity. These results support the dangers of inappropriate disposal of MSW to water bodies due to the high toxicity of the leachate produced highlighting the necessity of places of safe confinement and a treatment system more effective to it

Twenty years of Hendra virus: laboratory submission trends and risk factors for infection in horses

SUMMARY Hendra virus (HeV) was first described in 1994 in an outbreak of acute and highly lethal disease in horses and humans in Australia. Equine cases continue to be diagnosed periodically, yet the predisposing factors for infection remain unclear. We undertook an analysis of equine submissions tested for HeV by the Queensland government veterinary reference laboratory over a 20-year period to identify and investigate any patterns. We found a marked increase in testing from July 2008, primarily reflecting a broadening of the HeV clinical case definition. Peaks in submissions for testing, and visitations to the Government HeV website, were associated with reported equine incidents. Significantly differing between-year HeV detection rates in north and south Queensland suggest a fundamental difference in risk exposure between the two regions. The statistical association between HeV detection and stockhorse type may suggest that husbandry is a more important risk determinant than breed per se. The detection of HeV in horses with neither neurological nor respiratory signs poses a risk management challenge for attending veterinarians and laboratory staff, reinforcing animal health authority recommendations that appropriate risk management strategies be employed for all sick horses, and by anyone handling sick horses or associated biological samples.

Insecticidal activity of a protein extracted from bulbs of Phycella australis Ravenna against the aphids Acyrthosiphon pisum Harris and Myzus persicae Sulzer

Aphids cause significant losses in many agricultural crops and in many cases cause repeated insecticide sprays, which increase the risk of resistance. Therefore, other alternatives are needed to control them. The toxic, antireproductive, and feeding deterrent effects of a mannosebinding lectin isolated from bulbs of Phycella australis Ravenna (Amaryllidaceae), named Phycella australis agglutinin (PAA) was assayed on nymphs of the aphids Acyrthosiphon pisum Harris and Myzus persicae Sulzer fed with an artificial diet. After 72 h of PAA exposure, lethal concentration (LC50) values were 109 and 313 μg mL-1 for A. pisum and M. persicae, respectively, while LC90 values were 248 and 634 μg mL-1. Sub-lethal concentrations of PAA significantly reduced the aphid fecundity at a concentration of 80 μg mL-1. Only a total of 5.7 descendants per female were recorded for A. pisum (32% control progeny) and 12.4 for M. persicae (39% control progeny). Acyrthosiphon pisum was strongly deterred by PAA under choice conditions, as after 72 h exposed to 80 μg PAA mL-1 of diet, the feeding deterrent index was 0.91 for A. pisum and only 0.38 for M. persicae. In conclusion, the mannosebinding lectin isolated from bulbs of P. australis showed acute and chronical insecticidal activity against the pea and green peach aphids.

Adaptive Mechanisms of an Estuarine Synechococcus based on Genomics, Transcriptomics, and Proteomics

Picocyanobacteria are important phytoplankton and primary producers in the ocean. Although extensive work has been conducted for picocyanobacteria (i.e. Synechococcus and Prochlorococcus) in coastal and oceanic waters, little is known about those found in estuaries like the Chesapeake Bay. Synechococcus CB0101, an estuarine isolate, is more tolerant to shifts in temperature, salinity, and metal toxicity than coastal and oceanic Synechococcus strains, WH7803 and WH7805. Further, CB0101 has a greater sensitivity to high light intensity, likely due to its adaptation to low light environments. A complete and annotated genome sequence of CB0101 was completed to explore its genetic capacity and to serve as a basis for further molecular analysis. Comparative genomics between CB0101, WH7803, and WH7805 show that CB0101 contains more genes involved in regulation, sensing, and stress response. At the transcript and protein level, CB0101 regulates its metabolic pathways, transport systems, and sensing mechanisms when nitrate and phosphate are limited. Zinc toxicity led to oxidative stress and a global down regulation of photosystems and the translation machinery. From the stress response studies seven chromosomal toxin-antitoxin (TA) genes, were identified in CB0101, which led to the discovery of TA genes in several marine Synechococcus strains. The activation of the relB2/relE1 TA system allows CB0101 to arrest its growth under stressful conditions, but the growth arrest is reversible, once the stressful environment dissipates. The genome of CB0101 contains a relatively large number of genomic island (GI) genes compared to known marine Synechococcus genomes. Interestingly, a massive shutdown (255 out of 343) of GI genes occurred after CB0101 was infected by a lytic phage. On the other hand, phage-encoded host-like proteins (hli, psbA, ThyX) were highly expressed upon phage infection. This research provides new evidence that estuarine Synechococcus like CB0101 have inherited unique genetic machinery, which allows them to be versatile in the estuarine environment.

Micro and nanosystems for delivering local anesthetics

Introduction: One of the most common strategies for pain control during and after surgical procedures is the use of local anesthetics. Prolonged analgesia can be safely achieved with drug delivery systems suitably chosen for each local anesthetic agent.Areas covered: This review considers drug delivery formulations of local anesthetics designed to prolong the anesthetic effect and decrease toxicity. The topics comprise the main drug delivery carrier systems (liposomes, biopolymers, and cyclodextrins) for infiltrative administration of local anesthetics. A chronological review of the literature is presented, including details of formulations as well as the advantages and pitfalls of each carrier system. The review also highlights pharmacokinetic data on such formulations, and gives an overview of the clinical studies published so far concerning pain control in medicine and dentistry.Expert opinion: The design of novel drug delivery systems for local anesthetics must focus on how to achieve higher uploads of the anesthetic into the carrier, and how to sustain its release. This comprehensive review should be useful to provide the reader with the current state-of-art regarding drug delivery formulations for local anesthetics and their possible clinical applications.

Polyporus squamosus (Huds.) Fr from different origins: chemical characterization, screening of the bioactive properties and specific antimicrobial effects against Pseudomonas aeruginosa

Wild mushrooms are mainly collected during the rainy season and valued as a nutritious food and sources of natural medicines and nutraceuticals. The aim of this study was to determine the chemical composition and bioactive properties (antioxidant, antimicrobial and cytotoxicity) of Polyporus squamosus from two different origins, Portugal and Serbia. The sample from Portugal showed higher contents of as protein (17.14 g/100 g), fat (2.69 g/100 g), ash (3.15 g/100 g) and carbohydrates (77.02 g/100 g); the same sample gave the highest antioxidant activity: highest reducing power, DPPH radical scavenging activity, and lipid peroxidation inhibition in both β-carotene/linoleate and TBARS assay. These results could be related to its higher content in total tocopherols (1968.65 μg/100 g) and phenolic compounds (1.29 mg/100 g). Both extracts exhibited antibacterial activity against all the tested organisms. The samples from Serbia gave higher overall antibacterial activity and showed excellent antibiofilm activity (88.30 %). Overall, P. squamosus methanolic extracts possessed antioxidant, antimicrobial, antibiofilm and anti-quorum sensing activity, and without toxicity for liver cells. This investigation highlights alternatives to be explored for the treatment of bacterial infections, in particular against Pseudomonas aeruginosa. This study provides important results for the chemical and bioactive properties, especially antimicrobial activity of the mushroom P. squamosus. Moreover, to the authors’ knowledge this is the first report on sugars, organic acids, and individual phenolic compounds in P. squamosus.

Encapsulation of Local Anesthetic Bupivacaine in Biodegradable Poly(DL-lactide-co-glycolide) Nanospheres: Factorial Design, Characterization and Cytotoxicity Studies

Local anesthetic agents cause temporary blockade of nerve impulses productiong insensitivity to painful stimuli in the area supplied by that nerve. Bupivacaine (BVC) is an amide-type local anesthetic widely used in surgery and obstetrics for sustained peripheral and central nerve blockade. in this study, we prepared and characterized nanosphere formulations containing BVC. To achieve these goals, BVC loaded poly(DL-lactide-co-glycolide) (PLGA) nanospheres (NS) were prepared by nanopreciptation and characterized with regard to size distribution, drug loading and cytotoxicity assays. The 2(3-1) factorial experimental design was used to study the influence of three different independent variables on nanoparticle drug loading. BVC was assayed by HPLC, the particle size and zeta potential were determined by dynamic light scattering. BVC was determined using a combined ultrafiltration-centrifugation technique. The results of optimized formulations showed a narrow size distribution with a polydispersivity of 0.05%, an average diameter of 236.7 +/- 2.6 nm and the zeta potential -2.93 +/- 1,10 mV. In toxicity studies with fibroblast 3T3 cells, BVC loaded-PLGA-NS increased cell viability, in comparison with the effect produced by free BVC. In this way, BVC-loaded PLGA-NS decreased BVC toxicity. The development of BVC formulations in carriers such as nanospheres could offer the possibility of controlling drug delivery in biological systems, prolonging the anesthetic effect and reducing toxicity.

Bioaccumulation and effects of TBT on molluscs from southern Portugal

Tese de dout. Ciências e Tecnologias do Ambiente, Faculdade de Ciências do Mar e do Ambiente, Univ. do Algarve, 2004

Avaliação da ecotoxicidade de percolados em áreas de disposição de resíduos na região metropolitana de Natal/RN

Leachates are effluent produced by decomposition of solid waste, they have complex composition and can be highly toxic. Therefore such percolated liquid should be collected and treated properly to avoid environmental contamination of soil and of water bodies. The objective of this study was to evaluate the toxicity through ecotoxicological tests with Ceriodaphnia dubia (Cladocera - Crustacea) of percolated liquids generated in two different systems of municipal solid waste (MSW) disposal in the city of Natal/ RN: A Sanitary Landfill in the Metropolitan Region of Natal/ RN, and in a dump off area. Furthermore, it was evaluated the possible contamination of the underground water of the dump off area. Two monthly samples were taken at four points between the months of May/2009 and January/2010. The Point "A" corresponds to the end of the pond leachate treatment in ASRMN; The Point "B" corresponds to a containment pond at the dump. The Point "C" is an area near one of the cells of the dump off area where the leachate outcrops; The Point "D" stands for an underground water well at the area. The last point, called "E" was sampled only once and corresponds to the slurry produced by temporary accumulation of solid waste in the open area of the dump. The ecotoxicological tests, acute and chronic, followed the ABNT 13373/2005 rules, with some modifications. The samples were characterized by measuring the pH number, the dissolved oxygen (DO), the salinity, BOD5, COD, Cd, Cu, Pb, Cr, Fe, Mg, Ni, and Zn. At Point A, the average number of EC50-48h ranged between 1.0% and 2.77% (v/v), showing a high toxicity of the leachate to C.dubia in all months. To this point, positive correlations were found between the EC50- 48 with precipitation. Negative correlations were found between the EC50- 48h with salinity. At point B there was no response of the acute exposure of organisms to the test samples. At point C the EC50-48h ranged from 17.68% to 35.36% in just two months of the five ones analyzed, not correlated meaning. Point D, the EC50-48h level ranged between 12.31% and 71.27%, showed a negative correlation with, only, precipitation. Although it was observed toxicity of underground water in the Landfill Area, there was no evidence of water contamination by leachate, however, due to the toxic character of this water, additional tests should be conducted to confirm the quality of water that is used for human supply. At point E there was no acute toxicity. These results support the dangers of inappropriate disposal of MSW to water bodies due to the high toxicity of the leachate produced highlighting the necessity of places of safe confinement and a treatment system more effective to it

Assessment of the Occurrence and Potential Risks of Pharmaceuticals and their Metabolites in Fish and Water Using Liquid Chromatography Mass Spectrometry

A comprehensive method for the analysis of 11 target pharmaceuticals representing multiple therapeutic classes was developed for biological tissues (fish) and water. Water samples were extracted using solid phase extraction (SPE), while fish tissue homogenates were extracted using accelerated solvent extraction (ASE) followed by mixed-mode cation exchange SPE cleanup and analyzed by liquid chromatography tandem mass spectrometry (LC-MS/MS). Among the 11 target pharmaceuticals analyzed, trimethoprim, caffeine, sulfamethoxazole, diphenhydramine, diltiazem, carbamazepine, erythromycin and fluoxetine were consistently detected in reclaimed water. On the other hand, caffeine, diphenhydramine and carbamazepine were consistently detected in fish and surface water samples. In order to understand the uptake and depuration of pharmaceuticals as well as bioconcentration factors (BCFs) under the worst-case conditions, mosquito fish were exposed to reclaimed water under static-renewal for 7 days, followed by a 14-day depuration phase in clean water. Characterization of the exposure media revealed the presence of 26 pharmaceuticals while 5 pharmaceuticals including caffeine, diphenhydramine, diltiazem, carbamazepine, and ibuprofen were present in the organisms as early as 5 h from the start of the exposure. Liquid chromatography ultra-high resolution Orbitrap mass spectrometry was explored as a tool to identify and quantify phase II pharmaceutical metabolites in reclaimed water. The resulting data confirmed the presence of acetyl-sulfamethoxazole and sulfamethoxazole glucuronide in reclaimed water. To my knowledge, this is the first known report of sulfamethoxazole glucuronide surviving intact through wastewater treatment plants and occurring in environmental water samples. Finally, five bioaccumulative pharmaceuticals including caffeine, carbamazepine, diltiazem, diphenhydramine and ibuprofen detected in reclaimed water were investigated regarding the acute and chronic risks to aquatic organisms. The results indicated a low potential risk of carbamazepine even under the worst case exposure scenario. Given the dilution factors that affect environmental releases, the risk of exposure to carbamazepine will be even more reduced.

Real-time Biosensor for the Assessment of Nanotoxicity and Cancer Electrotherapy

Knowledge of cell electronics has led to their integration to medicine either by physically interfacing electronic devices with biological systems or by using electronics for both detection and characterization of biological materials. In this dissertation, an electrical impedance sensor (EIS) was used to measure the electrode surface impedance changes from cell samples of human and environmental toxicity of nanoscale materials in 2D and 3D cell culture models. The impedimetric response of human lung fibroblasts and rainbow trout gill epithelial cells when exposed to various nanomaterials was tested to determine their kinetic effects towards the cells and to demonstrate the biosensor’s ability to monitor nanotoxicity in real-time. Further, the EIS allowed rapid, real-time and multi-sample analysis creating a versatile, noninvasive tool that is able to provide quantitative information with respect to alteration in cellular function. We then extended the application of the unique capabilities of the EIS to do real-time analysis of cancer cell response to externally applied alternating electric fields at different intermediate frequencies and low-intensity. Decreases in the growth profiles of the ovarian and breast cancer cells were observed with the application of 200 and 100 kHz, respectively, indicating specific inhibitory effects on dividing cells in culture in contrast to the non-cancerous HUVECs and mammary epithelial cells. We then sought to enhance the effects of the electric field by altering the cancer cell’s electronegative membrane properties with HER2 antibody functionalized nanoparticles. An Annexin V/EthD-III assay and zeta potential were performed to determine the cell death mechanism indicating apoptosis and a decrease in zeta potential with the incorporation of the nanoparticles. With more negatively charged HER2-AuNPs attached to the cancer cell membrane, the decrease in membrane potential would thus leave the cells more vulnerable to the detrimental effects of the applied electric field due to the decrease in surface charge. Therefore, by altering the cell membrane potential, one could possibly control the fate of the cell. This whole cell-based biosensor will enhance our understanding of the responsiveness of cancer cells to electric field therapy and demonstrate potential therapeutic opportunities for electric field therapy in the treatment of cancer.