Combined effects of chemical and natural stressors to soil organisms and plants

Nas ��ltimas d��cadas, a Terra tem experimentado um aquecimento global e mudan��as nos padr��es de precipita����o. Muitos estudos sobre a avalia����o de risco de agrot��xicos em organismos n��o-alvo foram realizados com base em protocolos padronizados, com condi����es abi��ticas controladas. Mas, em campo, os organismos s��o expostos a flutua����es de v��rios fatores ambientais, bem como a poluentes, que podem alterar os limites de toler��ncia dos organismos aos stressores naturais, bem como alterar a toxicidade ou biodisponibilidade do qu��mico em causa. Considerando isso, o principal objetivo deste trabalho foi o de avaliar de que modo e em que medida os fatores ambientais (temperatura, humidade do solo e radia����o UV) podem interagir uns com os outros ou afetar a toxicidade do carbaril para invertebrados do solo e plantas. Para isso, foram utilizadas quatro esp��cies padr��o: Folsomia candida, Eisenia andrei, Triticum aestivum e Brassica rapa, e simulados diferentes cen��rios clim��ticos, com v��rios par��metros letais e subletais analisados. A exposição combinada foi analisada utilizando, quando poss��vel, a ferramenta MIXTOX, com base no modelo de refer��ncia de ac����o independente (IA) e poss��veis desvios, assim como r��cios sinerg��sticos/antagon��sticos (a partir de valores de EC50/LC50), quando a dose-resposta de um dos stressores n��o foi obtida. Todos os fatores de stress aplicados isoladamente causaram efeitos significativos sobre as esp��cies testadas e sua exposição combinada com carbaril, apresentaram respostas diferenciadas: para as minhocas, a seca e temperaturas elevadas aumentaram os efeitos delet��rios do carbaril (sinergismo), enquanto o alagamento e temperaturas baixas diminu��ram sua toxicidade (antagonismo). Para os col��mbolos, o modelo IA mostrou ser uma boa ferramenta para prever a toxicidade do carbaril tanto para temperaturas altas como para as baixas. Para as duas esp��cies de plantas foram encontradas diferen��as significativas entre elas: em termos gerais, as intera����es entre carbaril e os stressores naturais foram observadas, com sinergismo aparecendo como o padr��o principal relacionado com a radia����o UV, solos secos e temperaturas elevadas, enquanto o padr��o principal relacionado com temperaturas baixas e stress de alagamento foi o antagonismo. Quando os efeitos de dois stressores naturais (radia����o UV e humidade do solo) em plantas foram avaliados, uma intera����o significativa foi encontrada: a seca aliviou o efeito delet��rio da radia����o UV em T. aestivum e o alagamento aumentou os seus efeitos, mas para B. rapa a adi����o de ambos os stresses de ��gua causou um aumento (sinergismo) dos efeitos delet��rios da radia����o UV para todos os par��metros avaliados. Portanto �� necess��rio que as diferen��as sazonais e latitudinais, bem como as mudan��as clim��ticas globais, sejam integradas na avalia����o de risco de contaminantes do solo.

Oxidation and glycoxidation of phosphatidylserine and their effect in immune response : a lipidomic approach

Phosphatidylserine (PS) is a member of the class of phospholipids, and is distributed among all cells of mammalians, playing important roles in diverse biological processes, including blood clotting and apoptosis. When externalized, PS is a ligand that is recognized on apoptotic cells. It has been considered that before externalization PS is oxidized and oxPS enhance the recognition by macrophages receptors, however the knowledge about oxidation of PS is still limited. PS, like others phospholipids, has two fatty acyl chains and one polar head group, in this case is the amino acid serine. The modifications in PS structure can occur by oxidation of the unsaturated fatty acyl chains and by glycation of the polar head group, due to free amine group, thus increasing the susceptibility to oxidative events. The main goal of this work was to characterize and identify oxidized and glycoxidized PS, contributing to the knowledge of the biological role of oxidation products of PS, as well as of glycated PS, in immune and inflammatory processes. To achieve this goal, PS standards (1-palmitoyl-2-oleoyl-sn-glycero-3-phospho- L-serine (POPS), 1,2-dipalmitoyl-sn-glycero-3-phospho-L-serine (DPPS), 1- palmitoyl-2-linoleoyl-sn-glycero-3-phospho-L-serine (PLPS) and 1-palmitoyl-2- arachidonoyl-sn-glycero-3-phospho-L-serine (PAPS)) and glycated PS (PAPS and POPS) were induced to oxidize in model systems, using different oxidant reagents: HO��� and 2,2'-azobis-2-methyl-propanimidamide dihydrochloride (AAPH) . The detailed structural characterization of the oxidative products was performed by ESI-MS and MS/MS coupled to separation techniques such as off line TLC-MS and on line LC-MS, in order to obtained better characterization of the larger number of PS and glycated PS oxidation products. The results obtained in this work allowed to identify several oxidation products of PS and glycated PS with modifications in unsaturated fatty acyl chain. Also, oxidation products formed due to structural changes in the serine polar head with formation of terminal acetamide, terminal hydroperoxyacetaldehyde.and terminal acetic acid (glycerophosphacetic acid, GPAA) were identified. The mass spectrometric specific fragmentation pathway of each type of oxidation product was determined using different mass spectrometry approaches. Based on the identified fragmentation pathways, targeted lipidomic analysis was performed to detect oxidation products modified in serine polar head in HaCaT cell line treated with AAPH. The GPAA was detected in HaCaT cells treated with AAPH to induce oxidative stress, thus confirming that modifications in PS polar head is possible to occur in biological systems. Furthermore, it was found that glycated PS species are more prone to oxidative modifications when compared with non glycated PS. During oxidation of glycated PS, besides the oxidation in acyl chains, new oxidation products due to oxidation of the glucose moiety were identified, including PS advanced glycation end products (PSAGES). To investigate if UVA oxidative stress exerted changes in the lipidome of melanoma cell lines, particularly in PS profile, a lipidomic analysis was performed. The lipid profile was obtained using HILIC-LC-MS and GC-MS analysis of the total lipid extracts obtained from human melanoma cell line (SKMEL- 28) after UVA irradiation at 0, 2 and 24 hours. The results did not showed significant differences in PS content. At molecular level, only PS (18:0:18:1) decreased at the moment of irradiation. The most significant changes in phospholipids content occurred in phosphatidylcholines (PC) and phosphatidylinositol (PI) classes, with an increase of mono-unsaturated fatty acid (MUFA), similarly as observed for the fatty acid analysis. Overall, these data indicate that the observed membrane lipid changes associated with lipogenesis after UVA exposure may be correlated with malignant transformations associated with cancer development and progression. Despite of UVA radiation is associated with oxidative damage, in this work was not possible observe oxidation phospholipids. The anti/pro-inflammatory properties of the oxidized PLPS (oxPLPS) versus non-oxidized PLPS were tested on LPS stimulated RAW 264.7 macrophages. The modulation of intracellular signaling pathways such as NF-kB and MAPK cascades by oxPLPS and PS was also examined in this study. The results obtained from evaluation of anti/pro-inflammatory properties showed that neither PLPS or oxPLPS species activated the macrophages. Moreover only oxidized PLS were found to significantly inhibit NO production and iNOS and il1�� gene transcription induced by LPS. The analysis at molecular level showed that this was the result of the attenuation of LPS-induced c-Jun-N-terminal kinase (JNK) and p65 NF-kB nuclear translocation. Overall these data suggest that oxPLPS, but not native PLPS, mitigates pro-inflammatory signaling in macrophages, contributing to containment of inflammation during apoptotic cell engulfment. The results obtained in this work provides new information on the modifications of PS, facilitating the identification of oxidized species in complex samples, namely under physiopathologic conditions and also contributes to a better understanding of the role of oxPS and PS in the inflammatory response, in the apoptotic process and other biological functions.

Effects of environmental factors on the toxicity of pesticides to zebrafish embryos

During the last century mean global temperatures have been increasing. According to the predictions, the temperature change is expected to exceed 1.5��C in this century and the warming is likely to continue. Freshwater ecosystems are among the most sensitive mainly due to changes in the hydrologic cycle and consequently changes in several physico-chemical parameters (e.g. pH, dissolved oxygen). Alterations in environmental parameters of freshwater systems are likely to affect distribution, morphology, physiology and richness of a wide range of species leading to important changes in ecosystem biodiversity and function. Moreover, they can also work as co-stressors in environments where organisms have already to cope with chemical contamination (such as pesticides), increasing the environmental risk due to potential interactions. Therefore, the objective of this work was to evaluate the effects of climate change related environmental parameters on the toxicity of pesticides to zebrafish embryos. The following environmental factors were studied: pH (3.0-12.0), dissolved oxygen level (0-8 mg/L) and UV radiation (0-500 mW/m2). The pesticides studied were the carbamate insecticide carbaryl and the benzimidazole fungicide carbendazim. Stressors were firstly tested separately in order to derive concentration- or intensity-response curves to further study the effects of binary combinations (environmental factors x pesticides) by applying mixture models. Characterization of zebrafish embryos response to environmental stress revealed that pH effects were fully established after 24 h of exposure and survival was only affected at pH values below 5 and above 10. Low oxygen levels also affected embryos development at concentrations below 4 mg/L (delay, heart rate decrease and edema), and at concentrations below 0.5 mg/L the survival was drastically reduced. Continuous exposure to UV radiation showed a strong time-dependent impact on embryos survival leading to 100% of mortality after 72 hours of exposure. The toxicity of pesticides carbaryl and carbendazim was characterized at several levels of biological organization including developmental, biochemical and behavioural allowing a mechanistic understanding of the effects and highlighting the usefulness of behavioural responses (locomotion) as a sensitive endpoint in ecotoxicology. Once the individual concentration response relationship of each stressor was established, a combined toxicity study was conducted to evaluate the effects of pH on the toxicity of carbaryl. We have shown that pH can modify the toxicity of the pesticide carbaryl. The conceptual model concentration addition allowed a precise prediction of the toxicity of the jointeffects of acid pH and carbaryl. Nevertheless, for alkaline condition both concepts failed in predicting the effects. Deviations to the model were however easy to explain as high pH values favour the hydrolysis of carbaryl with the consequent formation of the more toxic degradation product 1- naphtol. Although in the present study such explanatory process was easy to establish, for many other combinations the ���interactive��� nature is not so evident. In the context of the climate change few scenarios predict such increase in the pH of aquatic systems, however this was a first approach focused in the lethal effects only. In a second tier assessment effects at sublethal level would be sought and it is expectable that more subtle pH changes (more realistic in terms of climate changes scenarios) may have an effect at physiological and biochemical levels with possible long term consequences for the population fitness.

Physiology and metabolic profile of Moringa olifera and Eucalyptus globulus plants subjected to climate changes

As altera����es clim��ticas emergentes t��m um grande impacto no crescimento e desenvolvimento de esp��cies florestais, nomeadamente em esp��cies de valor industrial e medicinal, como �� o caso do eucalipto (Eucalyptus globulus) e da moringa (Moringa oleifera). Assim, �� urgente conhecer as respostas fisiol��gicas e entender as varia����es que ocorrem nos perfis metab��licos de esp��cies vegetais. Neste trabalho, plantas jovens de Eucalyptus globulus foram expostas a radia����o UVB (12kJ/m2) e foram avaliadas as respostas fisiol��gicas e o perfil metab��lico, um e onze dias ap��s a aplica����o da radia����o. A dose de UVB usada n��o afetou as rea����es fotoqu��micas nem as trocas gasosas, contudo ao n��vel do metabolismo do carbono (AST e amido) e do conte��do de pigmentos verificaram-se pequenas altera����es (AST e pigmentos). Atrav��s da an��lise do perfil metab��lico de E. globulus foram encontrados compostos vol��teis e semi-vol��teis pertencentes ��s fam��lias dos terpenos, sesquiterpenos e alde��dos. Em geral, os sesquiterpenos e os ��lcoois monoterp��nicos apresentaram uma tend��ncia para manter e, em alguns casos, diminuir com o stress, enquanto que o grupos dos alde��dos aumentou e os monoterpenos apresentaram um comportamento mais heterog��neo. O E. globulus mostrou ser uma esp��cie tolerante �� aplica����o da dose de UVB usada neste trabalho. Por outro lado, plantas jovens de M. oleifera foram expostas a d��fice h��drico (DH). Um grupo de plantas foi recolhido um dia ap��s o final da exposição e o outro grupo ap��s onze dias do final da exposição. Foi avaliado o perfil metab��lico desta esp��cie atrav��s de GC/MS. Os dados cromatogr��ficos indicaram que em condi����es de stress (DH(1) e DH(11)), as quantidades de compostos associados a vias prim��rias e secund��rias de defesa (como os alcanos, ��lcoois, ��cidos carbox��licos, ester��is, amino��cidos e a��ucares) sofreram algumas altera����es. As plantas analisadas 11 dias ap��s a remo����o do stress mostraram maiores varia����es do perfil de metabolitos. No entanto, tanto um como onze dias ap��s a remo����o do stress, as plantas apresentaram a forma����o de novos rebentos. Apesar do perfil de metabolitos ter sofrido algumas altera����es, por n��o se registarem casos de morte, conclui-se que as plantas de moringa mostraram ser tolerantes aos tratamentos aplicados.