Genotoxicity investigation of a cyanobacterial toxin, cylindrospermopsin

Cylindrospermopsin (CYN), a potent cyanobacterial hepatotoxin produced by Cylindrospermopsis raciborskii and other cyanobacteria, is regularly found in water supplies in many parts of the world, and has been associated with the intoxication of humans and livestock. In this study, Balb/c mice were injected via the intraperitoneal (IP) route with a single dose of 0.2 mg/kg CYN. Animals were sacrificed at 6, 12, 24, 48 and 72 It. DNA was isolated from the mouse livers, and examined for strand breakage by alkaline gel electrophoresis (pH 12). Significant DNA strand breakage was observed in the mouse liver exposed to CYN, suggesting that induction of DNA strand breakage is probably one of the key mechanisms for CYN genotoxicity. (C) 2002 Elsevier Science Ltd. All rights reserved.

Toxicological aspects of treatment to remove cyanobacterial toxins from drinking water determined using the heterozygous P53 transgenic mouse model

The presence of toxic cyanobacteria in drinking water reservoirs renders the need to develop treatment methods for the 'safe' removal of their associated toxins. Chlorine has been shown to successfully remove a range of cyanotoxins including microcystins, cylindrospermopsin and saxitoxins. Each cyanotoxin requires specific treatment parameters, particularly solution pH and free chlorine residual. However, currently there has not been any investigation into the toxicological effect of solutions treated for the removal of these cyanotoxins by chlorine. Using the P53(def) transgenic mouse model mate and female C57BL/6J hybrid mice were used to investigate potential cancer inducing effects from such oral dosing solutions. Both purified cyanotoxins and toxic cell-free extract cyanobacterial solutions were chlorinated and administered over 90 and 170 days (respectively) in drinking water. No increase in cancer was found in any treatment. The parent cyanotoxins, microcystins, cylindrospermopsin and saxitoxins were readily removed by chlorine. There was no significant increase in the disinfection byproducts trihalomethanes or haloacetic acids, levels found were well below guideline values. Histological examination identified no effect of treatment solutions except male mice treated with chlorinated cylindrospermopsin (as a cell free extract). In this instance 40% of males were found to have fatty vacuolation in their livers, cause unknown. It is recommended that further toxicology be undertaken on chlorinated cyanobacterial solutions, particularly for non-genotoxic carcinogenic compounds, for example the Tg. AC transgenic mouse model. (C) 2003 Elsevier Science Ltd. All rights reserved.

Accessing Planktothrix species diversity and associated toxins using quantitative real-time PCR in natural waters

Tese de Doutoramento em Biologia apresentada à Faculdade de Ciências da Universidade do Porto, 2015.

Cianobactérias e toxicidade: impacte na saúde pública em Portugal e no Brasil

Um aumento da concentração de nutrientes na água poderá desencadear fluorescências de cianobactérias (densidades >200 cel/mL). Sob determinadas condições as cianobactérias produzem toxinas responsáveis pelo envenenamento de animais e humanos. O objetivo deste estudo é relacionar a ocorrência de fluorescências toxicas em Portugal e no Brasil. Para tal, em 2005 e 2006 foi estudado o fitoplâncton em três reservatórios em Portugal (região sul) e dois no Brasil (Minas Gerais e Pará). Comparativamente foi verificado maior diversidade nos reservatórios portugueses, com dominância de cianobactérias em período de primavera/verão/outono, pertencentes a géneros produtores de hépato e neurotoxinas (Microcystis sp, Aphanizomenon sp, Oscillatoria sp e Planktothrix sp.). No Brasil observou-se dominância de cianobactérias ao longo de todo o ano, com presença de Microcystis aeruginosa, produtora de hepatotoxina. Conclui-se que os reservatórios estudados apresentam géneros produtores de toxinas, com risco para a saúde pública, sendo fundamental implementar medidas que contribuam para mitigar esta situação. - ABSTRACT - An increasing of nutrients in water can conduct to the development of cyanobacteria blooms (density>2000 cels/mL). Under specific conditions cyanobacteria produce toxins responsible for acute poisoning of animals and humans. The aim of this study is to describe toxic blooms in Portugal and Brazil. Therefore, phytoplankton from three Portuguese reservoirs (South region) and two from Brazil (Minas Gerais and Pará) were studied in 2005 and 2006. Portuguese reservoirs showed more diversity with dominance of hepatic and neurotoxin genera producers (Microcystis sp, Aphanizomenon sp, Oscillatoria sp e Planktothrix sp.) along spring/summer/autumn seasons. In Brazil dominance of cyanobacteria was observed all along the year with the presence of Microcystis aeruginosa hepatotoxic producer. The studied reservoirs present toxins producers’ genera, with risk for public health, being fundamental the implementation of mitigation measures to reverse this situation.

Toxicidade a cianobactérias: impacte potencial na saúde pública em populações de Portugal e Brasil

Como recurso natural fundamental à vida, a água e os ecossistemas aquáticos devem ser alvo de avaliação contínua, no que se reporta à sua qualidade física, química e biológica. Segundo a Organização Mundial de Saúde cerca de 1,1 biliões de pessoas estão impossibilitadas em aceder a qualquer tipo de água potável e, as populações residentes nas proximidades de rios, lagoas, e reservatórios utilizam estas águas para as suas necessidades de consumo, aumentando o risco de transmissão de doenças. Enquanto constituintes da comunidade fitoplanctónica, as cianobactérias são microrganismos procariotas, fotossintéticos, que obtêm os nutrientes diretamente da coluna de água e, um aumento da concentração de nutrientes (principalmente azoto e fósforo), associado a condições ambientais favoráveis, pode desencadear um crescimento rápido originando fluorescências. Sob determinadas condições as cianobactérias podem produzir toxinas existindo registos que evidenciam que fluorescências toxicas são responsáveis pelo envenenamento agudo e morte de animais e humanos pelo que, a água utilizada para consumo humano deverá ser regularmente monitorizada para este elemento biológico. O objetivo deste estudo é relacionar a ocorrência de fluorescências de cianobactérias (> 2000 cel/ml) e toxicidade associada, com o impacte potencial na Saúde Pública avaliado através do consumo direto ou indireto da água. Em Portugal foram selecionados oito reservatórios situados na região Sul, pertencentes às bacias hidrográficas do Sado e Guadiana e estudados entre 2000 e 2008. No Brasil foram selecionados os reservatórios de Três Marias (Estado de Minas Gerais) e de Tucuruí (Estado do Pará) e estudados em 2005 e 2006 respetivamente. Os reservatórios foram caracterizados em termos físicos e químicos, tendo-se igualmente procedido à caracterização da comunidade fitoplanctónica através da identificação e quantificação dos principais grupos presentes em diferentes épocas do ano. Em termos fitoplanctónicos os reservatórios portugueses apresentaram maior diversidade,verificando-se contudo dominância das cianobactérias na comunidade. Associados a fluorescências, foram registados nestes reservatórios géneros produtores de hepato e neurotoxinas como Aphanizomenon sp, Microcystis aeruginosa e Oscillatoria sp. No Brasil, em situação de fluorescências, os géneros produtores de neuro e hepatotoxinas foram Microcystis (> 350.000 cels/ml) e Cylindrospermopsis. A presença destes géneros, poderá constituir um risco potencial para a saúde pública, pelo que é importante a implementação de medidas de mitigação em todos os reservatórios objeto de estudo, devendo essa atuação passar pelo controle do estado trófico no sentido de evitar o desenvolvimento de fluorescências. Assim sugere-se a implementação de um tratamento adequado para a produção de água de consumo e a organização de ações de sensibilização e aviso e informação às populações que utilizam os reservatórios em Portugal e no Brasil para diversos usos. - ABSTRACT - As a life fundamental natural resource, water and aquatic ecosystems must be continuously evaluated in their physical, chemical and biological quality. According World Health Organization, 1.1 billion people has no chance to access any kind of potable water. Populations living near rivers, lagoons or reservoirs use those waters to content their needs, increasing risks disease transmission. As members of phytoplankton community, cyanobacteria are prokaryotic, photosynthetic microorganisms and get its nutrients directly from water column. The increase of this nutrients (especially nitrogen and phosphorus) associated with favorable environment conditions, can support a sudden grow and instigate blooms. Under specific conditions cyanobacteria can produce toxins and several records have shown that toxic blooms are responsible by acute poisoning and death in animals and humans so, water for human consumption must be regularly surveyed for this biologic element. The aim of this study is to correlate Cyanobacteria blooms (>2.000cels/ml) and connected toxicity with public health impact, evaluated through water consumption. In Portugal, eight reservoirs located in the South region were selected and study between 2000 and 2008. In Brazil, Três Marias reservoir (Minas Gerais Provence) and Tucuruí (Pará Provence) were selected and study in 2005 and 2006. Reservoirs were characterized in physical and chemical aspects, as well as phytoplankton community, through identification and counting of main present groups along study period. In bloom circumstances, liver toxins and neurotoxins producers like Aphanizomenon sp, Microcystis aeruginosa and Oscillatoria sp. were founded in Portuguese reservoirs. In Brazil, cyanobacteria genera involved in toxic bloom were Microcystis (> 350.000 cels/ml) and Cylindrospermopsis. This genera presence represents a potential risk for public health, and show the requirement to implement mitigation measures in all study reservoirs. These measures can be represented by water eutrophication control to avoid blooms, by appropriate treatments of water to human consumption, and public warnings or information to dose people in Portugal and Brazil that use these reservoirs to several activities.

Mould and yeast identification in archival settings: preliminary results on the use of traditional methods and molecular biology options in Portuguese archives

This project was developed to fully assess the indoor air quality in archives and libraries from a fungal flora point of view. It uses classical methodologies such as traditional culture media – for the viable fungi – and modern molecular biology protocols, especially relevant to assess the non-viable fraction of the biological contaminants. Denaturing high-performance liquid chromatography (DHPLC) has emerged as an alternative to denaturing gradient gel electrophoresis (DGGE) and has already been applied to the study of a few bacterial communities. We propose the application of DHPLC to the study of fungal colonization on paper-based archive materials. This technology allows for the identification of each component of a mixture of fungi based on their genetic variation. In a highly complex mixture of microbial DNA this method can be used simply to study the population dynamics, and it also allows for sample fraction collection, which can, in many cases, be immediately sequenced, circumventing the need for cloning. Some examples of the methodological application are shown. Also applied is fragment length analysis for the study of mixed Candida samples. Both of these methods can later be applied in various fields, such as clinical and sand sample analysis. So far, the environmental analyses have been extremely useful to determine potentially pathogenic/toxinogenic fungi such as Stachybotrys sp., Aspergillus niger, Aspergillus fumigatus, and Fusarium sp. This work will hopefully lead to more accurate evaluation of environmental conditions for both human health and the preservation of documents.

Encapsulamento de monómeros da lenhina pela β-ciclodextrina: estudos químico e de biodegradação

Mestrado em Engenharia Química.Ramo Tecnologias de Protecção Ambiental

Biorremediação de solos contaminados com produtos petrolíferos

Mestrado em Engenharia Química. Ramo Tecnologias de Protecção Ambiental.

Deteção do potencial cianotóxico em águas superficiais do Arquipélago dos Açores através de métodos moleculares

Dissertação de Mestrado, Ciências Biomédicas, 12 de Fevereiro de 2016, Universidade dos Açores.

Sensors for the detection and quantification of bacterial contamination in water for human use

The deterioration of water quality by Cyanobacteria cause outbreaks and epidemics associated with harmful diseases in Humans and animals because of the toxins that they release. Microcystin-LR is one of the hepatotoxins most widely studied and the World Health Organization, recommend a maximum value of 1mgL 1 in drinking water. Highly specific recognition molecules, such as molecular imprinted polymers are developed to quantify microcystins in waters for human use and shown to be of great potential in the analysis of these kinds of samples. The obtained results were auspicious, the detection limit found, 1.5mgL 1, being of the same order of magnitude as the guideline limit recommended by the WHO. This technology is very promising because the sensors are stable and specific, and the technology is inexpensive and allows for rapid on-site monitoring.

Square-wave voltametric method for determination of molinate concentration in a biological process using a hanging mercury drop electrode

A square-wave voltammetric (SWV) method using a hanging mercury drop electrode (HMDE) has been developed for determination of the herbicide molinate in a biodegradation process. The method is based on controlled adsorptive accumulation of molinate for 10 s at a potential of -0.8 V versus AgCl/Ag. An anodic peak, due to oxidation of the adsorbed pesticide, was observed in the cyclic voltammogram at ca. -0.320 V versus AgCl/Ag; a very small cathodic peak was also detected. The SWV calibration plot was established to be linear in the range 5.0x10-6 to 9.0x10-6 mol L-1; this corresponded to a detection limit of 3.5x10-8 mol L-1. This electroanalytical method was used to monitor the decrease of molinate concentration in river waters along a biodegradation process using a bacterial mixed culture. The results achieved with this voltammetric method were compared with those obtained by use of a chromatographic method (HPLC–UV) and no significant statistical differences were observed.

Dual augmentation for aerobic bioremediation of MTBE and TCE pollution in heavy metal-contaminated soil

In this work we isolated from soil and characterized several bacterial strains capable of either resisting high concentrations of heavy metals (Cd2+ or Hg2+ or Pb2+) or degrading the common soil and groundwater pollutants MTBE (methyl-tertbutyl ether) or TCE (trichloroethylene). We then used soil microcosms exposed to MTBE (50 mg/l) or TCE (50 mg/l) in the presence of one heavy metal (Cd 10 ppm or Hg 5 ppm or Pb 50 or 100 ppm) and two bacterial isolates at a time, a degrader plus a metalresistant strain. Some of these two-membered consortia showed degradation efficiencies well higher (49–182% higher) than those expected under the conditions employed, demonstrating the occurrence of a synergetic relationship between the strains used. Our results show the efficacy of the dual augmentation strategy for MTBE and TCE bioremediation in the presence of heavy metals.

Natural waste materials containing chitin as adsorbents for textile dyestuffs: batch and continuous studies

In this work three natural waste materials containing chitin were used as adsorbents for textile dyestuffs, namely the Anodonta (Anodonta cygnea) shell, the Sepia (Sepia officinalis) and the Squid (Loligo vulgaris) pens. The selected dyestuffs were the Cibacron green T3G-E (CI reactive green 12), and the Solophenyl green BLE 155% (CI direct green 26), both from CIBA, commonly used in cellulosic fibres dyeing, the most used fibres in the textile industry. Batch equilibrium studies showed that the materials’ adsorption capacities increase after a simple and inexpensive chemical treatment, which increases their porosity and chitin relative content. Kinetic studies suggested the existence of a high internal resistance in both systems. Fixed bed column experiments performed showed an improvement in adsorbents’ behaviour after chemical treatment. However, in the column experiments, the biodegradation was the main mechanism of dyestuff removal, allowing the materials’ bioregeneration. The adsorption was strongly reduced by the pore clogging effect of the biomass. The deproteinised Squid pen (grain size 0.500–1.41 mm) is the adsorbent with highest adsorption capacity (0.27 and 0.037 g/g, respectively, for the reactive and direct dyestuffs, at 20ºC), followed by the demineralised Sepia pen and Anodonta shell, behaving like pure chitin in all experiments, but showing inferior performances than the granular activated carbon tested in the column experiments.

Antitumor activity of hierridin B, a cyanobacterial secondary metabolite found in both filamentous and unicellular marine strains

Cyanobacteria are widely recognized as a valuable source of bioactive metabolites. The majority of such compounds have been isolated from so-called complex cyanobacteria, such as filamentous or colonial forms, which usually display a larger number of biosynthetic gene clusters in their genomes, when compared to free-living unicellular forms. Nevertheless, picocyanobacteria are also known to have potential to produce bioactive natural products. Here, we report the isolation of hierridin B from the marine picocyanobacterium Cyanobium sp. LEGE 06113. This compound had previously been isolated from the filamentous epiphytic cyanobacterium Phormidium ectocarpi SAG 60.90, and had been shown to possess antiplasmodial activity. A phylogenetic analysis of the 16S rRNA gene from both strains confirmed that these cyanobacteria derive from different evolutionary lineages. We further investigated the biological activity of hierridin B, and tested its cytotoxicity towards a panel of human cancer cell lines; it showed selective cytotoxicity towards HT-29 colon adenocarcinoma cells.