Relación estructura-función de la enzima uroporfirinógeno descarboxilasa: implicancias para la comprensión y el tratamiento de la porfiria cutánea tarda / Structure-function relationship of the enzyme uroporphyrinogen decarboxylase: implications for the understanding and treatment of porphyria cutanea tarda / Relação estrutura-função da enzima uroporfirinogênio descarbioxilase: implicações para a compreensão e tratamento da porfiria cutânea tardia / Reconocimiento a la trayectoria del Prof. Dr. Juan Miguel Castagnino†

Resumen La uroporfirinógeno descarboxilasa humana (UROD-h) es la quinta enzima del camino biosintético del hemo y su actividad deficiente, relacionada a mutaciones en su gen, se encuentra asociada a un subgrupo de porfirias. El objetivo de este trabajo fue estudiar la relación entre la dimerización de la enzima y su actividad enzimática y comprobar si la dimerización de UROD-h es imprescindible tanto para la primera etapa de la reacción (urogen→heptagen), como para la segunda etapa (heptagen→coprogen). Con ese objetivo, se expresó y purificó la UROD-h hasta homogeneidad, se analizó el comportamiento dímero-monómero bajo distintas condiciones que pudieran desplazar el equilibrio de dimerización y se evaluó la actividad enzimática en dichas condiciones. Los resultados obtenidos sugieren que la especie activa para la primera etapa de la reacción es el homodímero y que tanto el dímero como el monómero se comportan como especies activas para la segunda etapa de la reacción. Se propone que mutaciones clínicas como la Y311C, existentes en pacientes con porfiria cutánea tarda, podrían afectar la estabilidad del dímero y podrían ser el blanco para futuras terapias génicas.

Abstract Human uroporphyrinogen decarboxylase (UROD-h) is the fifth enzyme in the heme biosynthetic pathway and its deficient activity, related to mutations in its gene, is associated with a subset of porphyrias. The objective of this work was to study the relationship between the dimerisation of the enzyme and its enzymatic activity and to verify if the dimerisation of UROD-h is essential both for the first stage of the reaction (urogen→heptagen), and for the second stage (heptagen→ coprogen). With this objective, the UROD-h was expressed and purified to homogeneity, the dimer- monomer behaviour was analysed under different conditions, which could shift the dimerisation equilibrium, and the enzymatic activity was evaluated under these conditions. The results obtained suggest that the active species for the first stage of the reaction is the homodimer, and both the dimer and the monomer behaved as active species for the second stage of the reaction. It is proposed that clinical mutations such as Y311C, existing in porphyria cutanea tarda patients, could affect dimer stability and could be the target of future gene therapies.

Resumo A enzima uroporfirinogênio descarboxilase humana (UROD-h) é a quinta enzima da via biossintética do heme e sua atividade deficiente, relacionada com mutações em seu gene, está associada a um subgrupo de porfirias. O objetivo deste trabalho foi estudar a relação entre a dimerização da enzima e sua atividade enzimática e comprovar se a dimerização da UROD-h é imprescindível tanto para a primeira etapa da reação (urogênio→heptagênio), quanto para a segunda etapa (heptagênio→coprogênio). Com esse objetivo, a UROD-h foi expressa e purificada até a homogeneidade, o comportamento de dímero-monômero foi analisado sob diversas condições, que puderam deslocar o equilíbrio de dimerização, e a atividade enzimática foi avaliada em tais condições. Os resultados obtidos sugerem que a espécie ativa para a primeira etapa da reação é o homodímero, e tanto o dímero quanto o monômero se comportam como espécies ativas para a segunda etapa da reação. Propõe-se que mutações clínicas como Y311C, existentes em pacientes com porfiria cutânea tardia, poderiam afetar a estabilidade do dímero e poderiam ser o alvo de futuras terapias gênicas em porfiria cutânea tardia.

Individual and joint effects of glyphosate and cypermethrin formulations on two human cell lines.

The final effect of pesticides and their mixtures on living organisms is determined by the particular toxicodynamics of the system. Oxidative stress is one of the most studied molecular mechanisms of toxicity due to increasing evidence supporting its association with the toxic effects of different agrochemicals. In the present study we evaluated the presence of redox balance alterations in the cell lines HEp-2 and A549 exposed to formulations of glyphosate (March®) and cypermethrin (Superfina®) used separately or in combination (in a proportion equivalent to that used in soybean fields). We determined the activity of catalase, superoxide dismutase, glutathione S-transferase, intracellular GSH content, content of oxidized proteins (as measure of damage) and intracellular ROS content in both cell lines at two different mixture concentrations. Additionally, we evaluated the presence of statistical interaction to determine if the effect of the mixture on the parameters evaluated was additive, synergistic, or antagonistic. For this purpose, we used the Combination Subthresholding, Cooperative Effect and Statistical Linear Interaction approaches. We found that the interaction between pesticides depended on their concentration and the cellular models studied.

Toxicological effects of active and inert ingredients of imazethapyr formulation Verosil® against Scenedesmus vacuolatus (Chlorophyta).

Imazethapyr, a selective systemic herbicide, is widely used in agriculture and it is frequently detected in water bodies close to application areas. Like other agrochemicals, imazethapyr is commercialized in formulations containing a mixture of additives that increase the effectiveness of the active ingredient. These complex mixtures may cause adverse effects on non-target primary producers, such as microalgae, when they reach freshwater bodies. The aim of this study was to assess the effects, separately, of the formulation Verosil®, the formulation additives, and technical-grade imazethapyr, in the acidic form or as ammonium salt, on the microalga Scenedesmus vacuolatus (Chlorophyta). Verosil®, formulation additives, and acid imazethapyr significantly inhibited the growth of S. vacuolatus (Verosil® > formulation additives > acid imazethapyr) and caused morphological alterations from 2 mg L-1, 4 mg L-1, and 60 mg L-1 onwards, respectively. Verosil® and formulation additives caused the most adverse effect including membrane disorganization, cytoplasm contraction, cell wall thickening, thylakoidal membrane disaggregation, and starch granule accumulation. In addition, Verosil® and formulation additives increased the chl a/chl b ratio, indicating possible alterations in photosystems as a stress response. The carotene/chl a ratio was also increased in microalgae exposed to both Verosil® and formulation additives, suggesting an antioxidant response to these toxic compounds. All these results support the hypothesis that the formulation additives contribute significantly to the toxicity and alterations caused by the commercial formulation Verosil® on S. vacuolatus.

Integrated analysis of the quality of water bodies from the lower Paraná River basin with different productive uses by physicochemical and biological indicators.

The Paraná River basin is one of the most important in South America and is affected by human activities that take place on its margins. In particular, the De la Cruz stream flows through an industrial pole and the Arrecifes River goes mainly through agricultural fields. The aim of this study was to evaluate the water quality of the De la Cruz stream (S1) and the Arrecifes River (S2) by means of physicochemical parameters, including metals and pesticides concentrations. Since amphibians are good indicators of environmental quality, bioassays with Rhinella arenarum were carried on. For lethal and sublethal parameters, embryos and larvae were exposed to a dilution gradient of water samples and AMPHITOX Solution (AS) as negative control for 504 h. For the determination of oxidative stress biomarkers (Catalase -CAT-, Glutathione S-Transferase -GST-, Reduced Glutathione -GSH-, and lipid peroxidation -TBARS-), embryos and larvae were exposed to undiluted water samples and AS. For the determination of micronuclei, larvae at hind limb bud stage (S.28) were exposed to undiluted water samples, simultaneously with negative and positive controls (AS and cyclophosphamide 40 mg/L, respectively). Dissolved oxygen was low in both sites and the copper levels exceeded the Argentine limit for the protection of aquatic life. In embryos exposure, water sample from S1 caused lethal effects (504h-LC50 = 49 (28-71.6)%), increased TBARS levels, and GST and CAT activities. In larvae exposure, water sample from this site decreased CAT activity, while the water sample from S2 caused important lethal effects (504h-LC50 = 98.72 (60.60-302.52)%), low GSH levels and increased GST activity. Water samples from both sites induced higher micronuclei frequency than the negative control. This study alerts about the degradation of water quality of the studied sites including lethal and sublethal effects in R. arenarum that can jeopardize the native populations of this species.

Oxidative Response and Micronucleus Centromere Assay in HEp-2 Cells Exposed to Fungicide Iprodione.

The fungicide agents are a key component in the fruits and vegetables production. The Iprodione residues are one of the pesticide more frequently found in food products. The available data about the cytotoxicity of iprodione and its metabolites are scarce and do not allow characterization of its genotoxic potential and define the risk assessment.The human larynx epidermoid carcinoma cell line (HEp-2) has been shown to be sensitive to the toxic effects of xenobiotics of different origin and have been often used in citotoxicity and genotoxicity studies. The purpose of this paper is to evaluate the induction of genotoxicity and the role of oxidative stress in HEp-2cell line by exposure to the IP. The MTT test for viability resulted in CL50 85.86 (77.05-95.68) µg/mL of Iprodione. On the basis of this result, we proceeded to expose the cells to the sublethal concentrations (below the CL50) during 24 h to analyze the mitotic index and nuclear division index in order to determine the subcytotoxic concentrations of IP which the genotoxicity was evaluated. The subcytotoxic concentrations of 7, 17, and 25 µg/mL IP induced aneugenic effects as micronuclei centromere positive whereas 17 µg/mL was a threshold for centromere negative micronuclei induction in HEp-2 cells. The abnormal mitosis was induced for exposition of Hep-2 cells to the three concentrations. According to the result obtained, citotoxicity and genotoxicity oxidative stress studies were performed in 1.5, 7.0, and 25 µg/mL of IP. The results showed that the GSH intracellular content, the SOD activity and the levels of oxidative damage of the proteins were affected lead to redox imbalance. The decreased in the SOD activity and protein oxidation were in according to the result obtained to genotoxicity, suggesting that different biological targets could be affected.

Effect of glyphosate on the growth, morphology, ultrastructure and metabolism of Scenedesmus vacuolatus.

The effects of a commercial glyphosate formulation on the oxidative stress parameters and morphology (including the ultrastructure) of the phytoplanktonic green microalga Scenedesmus vacuolatus were evaluated. After 96 h of exposure to increasing herbicide concentrations (0, 4, 6, 8 mg L-1 active ingredient) with the addition of alkyl aryl polyglycol ether surfactant, the growth of the cultures decreased (96 h-IC50- 4.90 mg L-1) and metabolic and morphology alterations were observed. Significant increases in cellular volume (103-353%) and dry weight (105%) and a significant decrease in pigment content (41-48%) were detected. Oxidative stress parameters were significantly affected, showing an increase in the reactive oxygen species (ROS) and reduced glutathione (GSH) contents, oxidative damage to lipids and proteins and a decrease in the activities of the antioxidant enzymes superoxide dismutase (SOD) and catalase (CAT) and the detoxifying enzyme glutathione-S-transferase (GST). Cells exposed to glyphosate formulation were larger and showed an increase in vacuole size, bleaching, cell wall thickening and alteration of the stacking pattern of thylakoids. The results of this study showed the participation of oxidative stress in the mechanism of toxic action of the commercial glyphosate formulation on S. vacuolatus and the relation between the biochemical, morphological and ultrastructure alterations.

Corrigendum: Inducing the Alternative Oxidase Forms Part of the Molecular Strategy of Anoxic Survival in Freshwater Bivalves.

[This corrects the article on p. 100 in vol. 9, PMID: 29527172.].

Inducing the Alternative Oxidase Forms Part of the Molecular Strategy of Anoxic Survival in Freshwater Bivalves.

Hypoxia in freshwater ecosystems is spreading as a consequence of global change, including pollution and eutrophication. In the Patagonian Andes, a decline in precipitation causes reduced lake water volumes and stagnant conditions that limit oxygen transport and exacerbate hypoxia below the upper mixed layer. We analyzed the molecular and biochemical response of the North Patagonian bivalve Diplodon chilensis after 10 days of experimental anoxia (<0.2 mg O2/L), hypoxia (2 mg O2/L), and normoxia (9 mg O2/L). Specifically, we investigated the expression of an alternative oxidase (AOX) pathway assumed to shortcut the regular mitochondrial electron transport system (ETS) during metabolic rate depression (MRD) in hypoxia-tolerant invertebrates. Whereas, the AOX system was strongly upregulated during anoxia in gills, ETS activities and energy mobilization decreased [less transcription of glycogen phosphorylase (GlyP) and succinate dehydrogenase (SDH) in gills and mantle]. Accumulation of succinate and induction of malate dehydrogenase (MDH) activity could indicate activation of anaerobic mitochondrial pathways to support anoxic survival in D. chilensis. Oxidative stress [protein carbonylation, glutathione peroxidase (GPx) expression] and apoptotic intensity (caspase 3/7 activity) decreased, whereas an unfolded protein response (HSP90) was induced under anoxia. This is the first clear evidence of the concerted regulation of the AOX and ETS genes in a hypoxia-tolerant freshwater bivalve and yet another example that exposure to hypoxia and anoxia is not necessarily accompanied by oxidative stress in hypoxia-tolerant mollusks.

Cellular and molecular oxidative stress-related effects in uterine myometrial and trophoblast-decidual tissues after perigestational alcohol intake up to early mouse organogenesis.

The placenta plays a major role in embryo-fetal defects and intrauterine growth retardation after maternal alcohol consumption. Our aims were to determine the oxidative status and cellular and molecular oxidative stress effects on uterine myometrium and trophoblast-decidual tissue following perigestational alcohol intake at early organogenesis. CF-1 female mice were administered with 10% alcohol in drinking water for 17 days prior to and up to day 10 of gestation. Control females received ethanol-free water. Treated mice had smaller implantation sites compared to controls (p < 0.05), diminished maternal vascular lumen, and irregular/discontinuous endothelium of decidual vessels. The trophoblast giant cell layer was disorganized and presented increased abnormal nuclear frequency. The myometrium of treated females had reduced nitrite content, increased superoxide dismutase activity, and reduced glutathione (GSH) content (p < 0.05). However, the trophoblast-decidual tissue of treated females had increased nitrite content (p < 0.05), increased GSH level (p < 0.001), increased thiobarbituric acid-reactive substance concentration (p < 0.001), higher 3-nitrotyrosine immunoreaction, and increased apoptotic index (p < 0.05) compared to controls. In summary, perigestational alcohol ingestion at organogenesis induced oxidative stress in the myometrium and trophoblast-decidual tissue, mainly affecting cells and macromolecules of trophoblast and decidual tissues around early organogenesis, in CF-1 mouse, and suggests that oxidative-induced abnormal early placental formation probably leads to risk of prematurity and fetal growth impairment at term.

Oxidative stress and cellular and tissue damage in organogenic outbred mouse embryos after moderate perigestational alcohol intake.

Perigestational alcohol consumption by CF-1 mouse, from before mating up to the period of embryo organogenesis, leads to retarded early embryo development and neural tube defects. Here, we addressed if perigestational alcohol ingestion up to Day 10 of pregnancy induces oxidative stress and changes in macromolecules and organ tissues of early organogenic embryos. Adult CF-1 female mice were administered 10% ethanol in their drinking water for 17 days prior to mating and until Day 10 of gestation, whereas control females were administered ethanol-free water. Our results demonstrated significantly reduced Catalase abundance and activity and increased glutathione content in the embryos of ethanol-treated females. The nitrite level was significantly reduced, but TBARS (thiobarbituric acid reactive substances) content, an index of lipid peroxidation, did not change. Embryos derived from ethanol-treated females also showed higher abundance of 3-nitrotyrosine (3-NT)-containing proteins in all tissues, compared to the control group. Apoptosis was significantly increased in the ectoderm and mesoderm, but not in the heart-although this organ did contain more cleaved Caspase-3-positive cardiomyocytes per area of ventricular myocardium than controls. In sum, moderate perigestational alcohol ingestion up to Day 10 of gestation in mice induces oxidative stress by altering radical nitrogen species and antioxidant enzymatic and non-enzymatic mechanisms in embryos. Further, generalized protein nitration, due to unbalanced nitric oxide levels associated with tissue-specific apoptosis, was detected in embryos, suggesting that oxidative mechanisms may play an important role in the perigestational alcohol-induced malformation of organogenic embryos exposed to ethanol.

Influence of the spray adjuvant on the toxicity effects of a glyphosate formulation.

In the present study, the influence of the spray adjuvant on the toxicity effects of a glyphosate formulation was examined in HEp-2 cell line. We determined the median lethal concentration (LC50) of Atanor® (glyphosate formulation), Impacto® (spray adjuvant) and the mixture of both agrochemicals. We also compared the toxicities of the pesticides individually and in mixture and we analyzed the effects on oxidative balance from each treatment. Our results showed that all the agrochemicals assayed induce dose and time-dependent cytotoxicity and that the toxicity of Impacto® with Atanor® (mixture) was additive on HEp-2 cell line. All the agrochemicals assayed produced an increase in catalase activity and glutathione levels, while no effects were observed for superoxide dismutase and glutathione-S-transferase activities. We found an important increase in ROS production in cells treated with Atanor® and mixture. Besides, all the agrochemicals used triggered caspase 3/7 activation and hence induced apoptosis pathway in this cell line. In conclusion, our results demonstrated that the addition of adjuvant to glyphosate formulation increase the toxicity of the mixture in cell culture. Furthermore, cell culture exposed to agrochemical mixture showed an increased ROS production and antioxidant defenses.

Glyphosate commercial formulation causes cytotoxicity, oxidative effects, and apoptosis on human cells: differences with its active ingredient.

In the present study, the effects on oxidative balance and cellular end points of glyphosate, aminomethylphosphonic acid (AMPA), and a glyphosate formulation (G formulation) were examined in HepG2 cell line, at dilution levels far below agricultural recommendations. Our results show that G formulation had toxic effects while no effects were found with acid glyphosate and AMPA treatments. Glyphosate formulation exposure produced an increase in reactive oxygen species, nitrotyrosine formation, superoxide dismutase activity, and glutathione (GSH) levels, while no effects were observed for catalase and GSH-S-transferase activities. Also, G formulation triggered caspase 3/7 activation and hence induced apoptosis pathway in this cell line. Aminomethylphosphonic acid exposure produced an increase in GSH levels while no differences were observed in other antioxidant parameters. No effects were observed when the cells were exposed to acid glyphosate. These results confirm that G formulations have adjuvants working together with the active ingredient and causing toxic effects that are not seen with acid glyphosate.

Effects of sewage discharges on lipid and fatty acid composition of the Patagonian bivalve Diplodon chilensis.

Lipid and fatty acid (FA) composition and selected oxidative stress parameters of freshwater clams (Dipolodon chilensis), from a sewage-polluted (SMA) and a clean site, were compared. Trophic markers FA were analyzed in clams and sediment. Saturated FA (SAFA), and bacteria and sewage markers were abundant in SMA sediments, while diatom markers were 50% lower. Proportions of SAFA, branched FA, 20:5n-3 (EPA) and 22:6n-3 (DHA) were higher in SMA clams. Chronic exposure of D. chilensis to increasing eutrophication affected its lipid and FA composition. The increase in EPA and DHA proportions could be an adaptive response, which increases stress resistance but could also lead to higher susceptibility to lipid peroxidation TBARS, lipofuscins (20-fold) and GSH concentrations were higher in SMA clams. FA markers indicated terrestrial plant detritus and bacteria are important items in D. chilensis diet. Anthropogenic input in their food could be traced using specific FA as trophic markers.

Effect of seasonality on oxidative stress responses and metal accumulation in soft tissues of Aulacomya atra, a mussel from the South Atlantic Patagonian coast.

This study investigated the effects of pollution and its interaction with temperature on the oxidative status of the ribbed mussel Aulacomya atra in the southern Atlantic Patagonian coast. Animals were collected from four sites with different degree and type of human activity impact, during the summer and winter of 2011. Seawater chromium, copper, manganese, nickel and zinc concentrations were measured, as well as metal accumulation, lipid peroxidation, protein oxidation, reduced glutathione levels, and enzymatic activities of superoxide dismutase and glutathione-S-transferase in gills and digestive glands. Metal bioaccumulation and oxidative stress responses in both tissues were generally higher in mussels from harbor areas. Water temperature had a remarkable effect on gill SOD activity and protein oxidation during winter in mussels from all locations. Methodologically, we conclude that measuring both metal bioaccumulation and oxidative stress responses allowed for a more accurate assessment of the biological effects of metal present in seawater.

Cholinesterases and neurotoxicity as highly sensitive biomarkers for an organophosphate insecticide in a freshwater gastropod (Chilina gibbosa) with low sensitivity carboxylesterases.

In the Upper Valley of Río Negro and Río Neuquén in Argentina, agriculture represents the second most important economic activity. Azinphos-methyl has been found in water from this region throughout the year at a maximum concentration of 22.48 µg L(-1) during the application period. Toxicological studies on local non-target species have been performed mostly on vertebrates, while mollusks, which could be more sensitive, have not been studied so far. This work aims to characterize cholinesterase (ChE) and carboxilesterase (CE) activities of Chilina gibbosa, a freshwater gastropod native to southern Argentina and Chile. These enzymes, together with neurotoxicity signals, are evaluated herein after as sensitive biomarkers of exposure to azinphos-methyl at environmentally relevant concentrations. Effects of azinphos-methyl on antioxidant defenses: glutathione (GSH), catalase (CAT), superoxide dismutase (SOD) and glutathione S-transferase (GST) are also studied in order to complete a set of biomarkers with different sensitivity and specificity, to propose C. gibbosa as a sentinel species. The highest specific activity was obtained with acetylthiocholine as substrate, followed by propionylthiocholine (83% in comparison to acetylthiocholine) and butyrylthiocholine (19%).The lowest Km and the highest efficiency for ChE were obtained with acetylthiocholine. Regarding CEs activities, a higher efficiency was obtained with p-nitrophenyl butyrate than with p-nitrophenyl acetate. Eserine produced significant inhibition of ChE activity (81% with 0.001 mM and 98% with 1mM) while iso-OMPA did not produce any significant effect on ChE. Our results show that C. gibbosa ChE is very sensitive to azinphos-methyl (CI50 0.02 µg L(-1)) while CEs are inhibited at higher concentrations (CI50 1,000 µg L(-1)). CEs have been reported to be more sensitive to OPs than ChEs in most of the aquatic invertebrates protecting the organisms from neurotoxic effects. In contrast, C. gibbosa, has ChE which are much more sensitive to azinphos-methyl than CEs and shows marked signs of neurotoxicity. Regarding antioxidant defenses, GSH levels were significantly increased by 0.02 and 20 µg L(-1) azinphos-methyl (80 and 103%, respectively), CAT activity was increased 85% only at 0.02 µg L(-1) and SOD and GST did not show any significant response. Since ChE activity, neurotoxicity signs, GSH and CAT are sensitive biomarkers of acute exposure to azinphos-methyl at environmental concentrations C. gibbosa could be included as sentinel species in monitoring programs of pesticide hazard in regions of Argentina and Chile.

Estrés oxidativo en cefalópodos: I. Determinación de TBARS / Oxidative stress in cephalopods: I. TBARS determination

El estrés oxidativo se produce cuando se genera un desbalance desfavorable entre las especies reactivas del oxígeno y las defensas antioxidantes, provocando daño oxidativo a macromoléculas. Varios estudios han resaltado la importancia del estrés oxidativo en el campo de la ecotoxicología, particularmente su relación con el impacto que generan los contaminantes que alcanzan los cuerpos de agua. El cuantifcar los parámetros de estrés oxidativo ha permitido el uso de los mismos como herramienta de diagnóstico (biomarcadores), con capacidad predictiva del impacto de los contaminantes sobre los organismos. Uno de los índices más frecuentemente utilizados para estimar el daño oxidativo a lípidos es la determinación de sustancias reactivas al ácido tiobarbitúrico (TBARS), producto fnal de la peroxidación lipídica. Octopus tehuelchus es un importante recurso pesquero en la costa patagónica, expuesto en algunas áreas a contaminación antrópica. Dado que el estudio de parámetros de estrés oxidativo aún no ha sido abordado en esta Clase de moluscos y que en muchos modelos biológicos, los contaminantes ambientales actúan generando estrés oxidativo, es clave encontrar sus blancos de acción, para empezar a caracterizar las alteraciones metabólicas y fsiológicas asociadas a su mecanismo de acción. El objetivo de este trabajo fue la puesta a punto del método de determinación de daño oxidativo a lípidos en distintos tejidos del pulpo Octopus tehuelchus desde modelos previamente ensayados en el laboratorio.

Oxidative stress occurs when there is an unfavorable imbalance between reactive oxygen species and antioxidant defenses, causing oxidative damage to macromolecules. Several studies have highlighted the importance of oxidative stress in the ecology feld related to the impact generated by pollutants reaching water bodies. The quantifcation of oxidative stress parameters led to their use as diagnostic tools (biomarkers) with predictive capability of showing the impact of pollutants on organisms. One of the most frequently used indexes to estimate the oxidative damage to lipids is the determination of reactive thiobarbituric acid substances (TBARs) (fnal product of lipid peroxidation). Octopus tehuelchus is an important fshery resource in the Patagonian coast exposed to anthropogenic pollution. The study of oxidative stress parameters has not been yet tackled in this class of molluscs. Taking into account that, in many biological models, environmental pollutants generate oxidative stress, it is important to fnd their targets of action, to start to characterize metabolic and physiological alterations associated to their mechanisms of action. The aim of this work was to adjust the method of determination of oxidative damage to lipids in various tissues of the octopus, Octopus tehuelchus, from models previously tested in the laboratory.

Estrés oxidativo en cefalópodos: I. Determinación de TBARS / Oxidative stress in cephalopods: I. TBARS determination

El estrés oxidativo se produce cuando se genera un desbalance desfavorable entre las especies reactivas del oxígeno y las defensas antioxidantes, provocando daño oxidativo a macromoléculas. Varios estudios han resaltado la importancia del estrés oxidativo en el campo de la ecotoxicología, particularmente su relación con el impacto que generan los contaminantes que alcanzan los cuerpos de agua. El cuantifcar los parámetros de estrés oxidativo ha permitido el uso de los mismos como herramienta de diagnóstico (biomarcadores), con capacidad predictiva del impacto de los contaminantes sobre los organismos. Uno de los índices más frecuentemente utilizados para estimar el daño oxidativo a lípidos es la determinación de sustancias reactivas al ácido tiobarbitúrico (TBARS), producto fnal de la peroxidación lipídica. Octopus tehuelchus es un importante recurso pesquero en la costa patagónica, expuesto en algunas áreas a contaminación antrópica. Dado que el estudio de parámetros de estrés oxidativo aún no ha sido abordado en esta Clase de moluscos y que en muchos modelos biológicos, los contaminantes ambientales actúan generando estrés oxidativo, es clave encontrar sus blancos de acción, para empezar a caracterizar las alteraciones metabólicas y fsiológicas asociadas a su mecanismo de acción. El objetivo de este trabajo fue la puesta a punto del método de determinación de daño oxidativo a lípidos en distintos tejidos del pulpo Octopus tehuelchus desde modelos previamente ensayados en el laboratorio.(AU)

Oxidative stress occurs when there is an unfavorable imbalance between reactive oxygen species and antioxidant defenses, causing oxidative damage to macromolecules. Several studies have highlighted the importance of oxidative stress in the ecology feld related to the impact generated by pollutants reaching water bodies. The quantifcation of oxidative stress parameters led to their use as diagnostic tools (biomarkers) with predictive capability of showing the impact of pollutants on organisms. One of the most frequently used indexes to estimate the oxidative damage to lipids is the determination of reactive thiobarbituric acid substances (TBARs) (fnal product of lipid peroxidation). Octopus tehuelchus is an important fshery resource in the Patagonian coast exposed to anthropogenic pollution. The study of oxidative stress parameters has not been yet tackled in this class of molluscs. Taking into account that, in many biological models, environmental pollutants generate oxidative stress, it is important to fnd their targets of action, to start to characterize metabolic and physiological alterations associated to their mechanisms of action. The aim of this work was to adjust the method of determination of oxidative damage to lipids in various tissues of the octopus, Octopus tehuelchus, from models previously tested in the laboratory.(AU)

Direct and indirect effects of the glyphosate formulation Glifosato Atanor® on freshwater microbial communities.

Glyphosate-based formulations are among the most widely used herbicides in the world. The effect of the formulation Glifosato Atanor(®) on freshwater microbial communities (phytoplankton, bacterioplankton, periphyton and zooplankton) was assessed through a manipulative experiment using six small outdoor microcosms of small volume. Three of the microcosms were added with 3.5 mg l(-1) of glyphosate whereas the other three were left as controls without the herbicide. The treated microcosms showed a significant increase in total phosphorus, not fully explained by the glyphosate present in the Glifosato Atanor(®). Therefore, part of the phosphorus should have come from the surfactants of the formulation. The results showed significant direct and indirect effects of Glifosato Atanor(®) on the microbial communities. A single application of the herbicide caused a fast increase both in the abundance of bacterioplankton and planktonic picocyanobacteria and in chlorophyll a concentration in the water column. Although metabolic alterations related to oxidative stress were induced in the periphyton community, the herbicide favored its development, with a large contribution of filamentous algae typical of nutrient-rich systems, with shallow and calm waters. An indirect effect of the herbicide on the zooplankton was observed due to the increase in the abundance of the rotifer Lecane spp. as a consequence of the improved food availability given by picocyanobacteria and bacteria. The formulation affected directly a fraction of copepods as a target. It was concluded that the Glifosato Atanor(®) accelerates the deterioration of the water quality, especially when considering small-volume water systems.

Oxidative damage and antioxidant response of Allium cepa meristematic and elongation cells exposed to metronidazole.

The toxicity of metronidazole (MTZ) in meristematic and elongation zones of Allium cepa roots was analyzed for 30 h of exposition. Toxic effects were evaluated by lipid peroxidation (content of thiobarbituric-reactive substances [TBARS]), reduced glutathione (GSH) levels, ascorbate acid and dehydroascorbate acid content, and enzymatic activities of superoxide dismutase and catalase. The root zones showed differentiated susceptibility to MTZ. In the elongation zone, MTZ induced an increase of TBARS content and a significant rise in GSH levels, whereas in the meristematic zone, lipid peroxidation was not observed and all antioxidant defense parameters analyzed were significantly increased. These results indicate that MTZ exposure induced oxidative stress in A. cepa roots, and that the antioxidant defenses in the meristematic zone are more efficient compared with the elongation zone, which is probably related to higher oxidative metabolism of meristematic tissue.

Oxidative stress and histological alterations produced by dietary copper in the fresh water bivalve Diplodon chilensis.

The aim of this work was to study the oxidative stress effects and histological alterations caused by dietary copper on the filter-feeding freshwater mussel Diplodon chilensis. Bivalves were fed during 6 weeks with the green algae Scenedesmus vacuolatus previously exposed to copper. Metal concentration in algae cultures and bivalve digestive gland was measured by TXRF. A maximum accumulation of 0.49 µg Cu/mg protein was detected at week 6. Also at this week, the hepatosomatic index (HSI) showed the highest decrease (50%) in response to Cu exposure. SOD and GST activities were significantly increased at weeks 4, 5 and 6, reaching an activity on average 50% higher than in controls for GST. CAT activity and GSH increased significantly at weeks 5 and 6. Despite this response, oxidative damage measured as TBARS and carbonyl groups contents increased significantly at weeks 4, 5 and 6, respectively. Digestive tubule and duct atrophy and cell-type replacement in treated mussels were observed by histological studies. The presence of intracellular rhodanine-positive granules, suggests copper accumulation in intracellular vacuoles of digestive cells.