María del Carmen Ríos de Molina

Oxidative stress and antioxidant defenses in two green microalgae exposed to copper

The aim of this work was to assess the effects of 1 week copper exposure (6.2, 108, 210 and 414microM) on Scenedesmus vacuolatus and Chlorella kessleri. The strains showed different susceptibility to copper. Copper content was determined in both strains by total X-ray reflection fluorescence analysis (TXRF). In S. vacuolatus, the increase of medium copper concentration induced an augmentation of protein and MDA content, and a significant decrease in the chlorophyll a/chlorophyll b ratio. S. vacuolatus showed a significant increase of catalase activity in 210 and 414microM of copper, and a significant increment of SOD activity and GSH content only in 414microM of copper. On the contrary, C. kessleri did not show significant differences in these parameters between 6.2 and 108microM of copper. Increased copper in the environment evokes oxidative stress and an increase in the antioxidant defenses of S. vacuolatus.

Thyroid function and thyroxine metabolism in hexachlorobenzene-induced porphyria

The effects of hexachlorobenzene (HCB) administration on the development of porphyria and on changes in thyroid function and thyroid hormone metabolism were examined. Female Wistar rats were treated with HCB for 1 or 8 weeks. At both treatment times liver weight was notably increased with a slight change in thyroid weight at 8 weeks. Serum thyroxine (T4) levels were depressed, whereas levels of triiodothyronine (T3) were not depressed significantly at both treatment times. One or eight weeks of HCB treatment did not alter the incorporation and distribution of [125I] into intrathyroidal aminoacids. A 50% reduction in protein bound iodine (PB[125I]) was seen in both groups of animals. HCB altered [125I]T4 metabolism in rat liver slices, increasing T4 dehalogenation. HCB administration for 1 week did not affect urinary excretion of porphyrins or their precursors, or hepatic porphyrin content. The activity of aminolaevulinate synthase was not affected, but there was a 25% and 51% inhibition in porphyrinogen carboxy-lyase (PCL) activity for the uroporphyrinogen disappearance or the coproporphyrinogen formation respectively. After 8 weeks of HCB administration the rats showed a characteristic porphyria. Our results show that HCB treatment increased hepatic thyroxine metabolism, without alterations in thyroid hormone synthesis. Serum T4 and PCL activity behaved differently in both time- and dose-dependent studies, with serum T4 being the more sensitive parameter which responded at earlier times and lower doses.

Investigations on the presence of porphyrinogen carboxy-lyase inhibitor in the liver of rats intoxicated with hexachlorobenzene

Abstract 1. 1. It has been reported that hexachlorobenzene (HCB) in vivo produces hepatic porphyrinogen carboxy-lyase (PCL) decrease and pentachlorophenol (PCP) is one of its metabolites. In order to investigate if such decrease is due to an enzyme inhibitor present in the porphyric livers, the effects of the following additions on the normal rat liver PCL activity were investigated: (a) a porphyric liver preparation (free of porphyrins), (b) a heat deproteinized porphyric liver preparation, (c) HCB, PCP, several chloro and nitrophenols and other related compounds. 2. 2. The results of (a) and (b) would indicate that in porphyric liver there is probably an inhibitor tightly bound to the “porphyric” enzyme which is not separated by Sephadex G-25 and that produces an inhibition of “normal” enzyme smaller than that observed in vivo for HCB, the difference being perhaps due to a protein synthesis level action. 3. 3. From the results of (c) it is observed that (i) there are several drugs that inhibit hepatic PCL in different degrees, PCP, tetrachlorophenol and 2,4-dinitrophenol being the most effective, while HCB has no effect at any of the assayed concentrations, (ii) the inhibitory effect of these compounds could be mainly due to the presence of phenolic group and increased by the presence of electrophilic groups in the benzene ring, (iii) the above mentioned inhibitor made evident by the heating assays could be PCP, HCB being discarded because the absence of effect. However, the marked inhibitory effect of PCP decreases and disappears at lower concentrations. 4. 4. The physiological role of PCP in the production mechanism of HCB porphyria is discussed.

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.

Ultrastructure and X-ray microanalysis of Euglena gracilis (Euglenophyta) under chromium stress

I. Rocchetta, P.I. Leonardi, G.M. Amado Filho, M. del Carmen Ríos de Molina and V. Conforti. 2007. Ultrastructure and X-ray microanalysis of Euglena gracilis (Euglenophyta) under chromium stress. Phycologia 46: 300–306. DOI: 10.2216/06-49.1 The effect of chromium on the biology of Euglena gracilis was studied. The ultrastructural modifications caused by this metal and its location within the cell were analyzed by TEM and EDXA, respectively. The effects of chromium on protein, pigment, and lipid contents were also studied in order to evaluate the metabolic responses to metal exposure. Two strains of Euglena gracilis, UTEX 753 (from the Culture Collection of Texas University) and MAT (isolated from the Matanza River), were used in this research. Both were grown in photoauxotrophic and photoheterotrophic conditions and exposed to different metal concentrations. In all treated cells, increases in total protein and lipid contents, changes in chlorophyll amount, and alterations in fine structure were observed, especially with the higher concentration tested. In photosynthetic treated cells, assays showed chloroplast thylakoid disorganization, the presence of cytoplasm lipid globules, and several vacuoles with electron-dense inclusions and remnants of membranes inside. Nuclei presented lobulations, and eventually total fragmentation in some cells treated with the highest chromium concentration was seen, suggesting that chromium cytotoxicity leads to cellular death. The EDXA spectrum showed well-defined Cr and S peaks in the vacuoles containing electron-dense inclusions and remnants of membranes from autotrophic MAT samples. These results indicate that the different defense mechanisms against chromium depend on strain type and culture conditions. The S peak detected in MAT would suggest that sulfur-rich proteins groups play an important role in the detoxification system inducing metal-complex accumulation into vacuoles.

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.

How does hexachlorobenzene treatment affect liver uroporphyrinogen decarboxylase

The aims of the present work were: (1) to investigate whether the strong decrease of liver uroporphyrinogen decarboxylase (UroD) activity observed in experimental porphyria cutanea tarda is due to alteration of the enzymatic protein and (2) to improve the knowledge about the normal liver enzyme. With these purposes, several physicochemical studies for enzymatic characterization were carried out comparatively on the 12-fold purified liver enzyme of both normal and hexachlorobenzene porphyric rat. The study shows that the enzyme from porphyric rats has a higher activation energy, lower reactivity index and lower optimum pH than the normal one. In addition, it did not reach the Vmax at any of the substrate concentrations assayed (up to 28 microM uroporphyrinogen III), while the normal enzyme reached the plateau around 14 microM. The porphyric enzyme appears to be more protected than the normal against the inhibitory action of several metals, particularly Cu2+ and Pb2+, and against thermal inactivation. Zn2+ did not affect enzymatic activity, whereas Cu2+, Hg2+, Fe2+, Pb2+, and Cd2+ lowered the activities of both normal and porphyric enzyme in a dose-related way. It was also observed that the larger the atomic radius in its hydrated state, the lower the effect of the metal. Neither glutathione nor dithiothreitol significantly altered enzymatic activity in the range of concentrations assayed. beta-Mercaptoethanol had diverse effects, as regards both the concentration assayed and the enzymatic sample used. Assays with cystine showed a dual behaviour of both normal and porphyric enzymatic activity. Western blots for both preparations revealed a single band (65 kDa) with a similar intensity. This study show that hexachlorobenzene treatment modifies the physicochemical properties of liver UroD leading to a sharp decrease of its activity, without affecting its antigenic reactivity probably as a consequence of changes at the conformational level promoted by the binding of its reported inhibitor.

Dietary copper effects in the estuarine crab, Neohelice (Chasmagnathus) granulata, maintained at two different salinities.

We analyzed the dietary copper effects in the estuarine crab Neohelice (Chasmagnathus) granulata and its interaction with water salinity. Crabs were maintained at 2 per thousand and 30 per thousand salinity for 5 weeks and they were fed with commercial food supplemented with the green alga Scenedesmus vacuolatus previously exposed to copper. No mortalities were observed, but crabs maintained at 2 per thousand salinity accumulated on average 40% more copper compared to animals maintained at 30 per thousand salinity. At 2 per thousand salinity, superoxide dismutase (SOD) activity and reduced glutathione (GSH) levels were increased at the first and second weeks, respectively, while lipid peroxidation and protein oxidation were evident after 4 weeks of copper exposure. At 30 per thousand salinity, all measured variables increased progressively but were significantly higher only at the end of the assay (5th week), except for protein oxidation that remained unchanged throughout the experiment. The hepatosomatic index (HSI) was significantly decreased in response to copper exposure, but only in crabs acclimated to 2 per thousand. These findings have suggested that dietary copper exposure induces greater metal accumulation and larger oxidative stress responses in crabs maintained at 2 per thousand salinity.

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.

A Parachlorella kessleri (Trebouxiophyceae, Chlorophyta) strain from an extremely acidic geothermal pond in Argentina

Juárez Á.B., Vélez C.G., Iñiguez A.R., Martínez D.E., Rodríguez M.C., Vigna M.S. and Ríos de Molina M.C. 2011. A Parachlorella kessleri (Trebouxiophyceae, Chlorophyta) strain from an extremely acidic geothermal pond in Argentina. Phycologia 50: 413–421. DOI: 10.2216/10-79.1 Acidic geothermal environments are rare and very few organisms can thrive in them. Chlorella or Chlorella-like species have been described in a variety of environments but their presence in extremely acidic waters have rarely been reported. A Chlorella-like alga (strain BAFC CA10) was isolated from Laguna Verde, a volcanic mesothermal acidic pond from northern Patagonia (Argentina). Applying a multimethod approach including microscopic, ultrastructural, chemical and phylogenetic analyses, the acidotolerant strain BAFC CA10 was identified as Parachlorella kessleri ( =  Chlorella kessleri) on the basis of comparisons with the authentic strain SAG 211-11g. Our results confirmed the initial characterization of the Laguna Verde strain as C. kessleri, but since this species was transferred to the new genus Parachlorella, strain BAFC CA10 should be designated as P. kessleri. Histochemical staining and sugar composition of the cell-wall amorphous and fibrillar matrix, together with transmission electron microscopy of the cells, showed the distinctive characters of the species: (1) glucosamine was the exclusive component of the fibrillar cell wall; (2) rhamnose, galactose, glucose and xylose together with minor quantities of arabinose, mannose and fucose were present in the cell-wall hemicellulose matrix; (3) there was a single parietal chloroplast surrounding the entire cell with a small aperture (“mantel-shaped”); (4) there was one pyrenoid in the thickening of the chloroplast surrounded by two starch granules and bisected by two thylakoids; and (5) the cell wall was an electron-transparent homogeneous structure 60–80 nm thick. Phylogenetic analyses of concatenated small-subunit and internal transcribed sequence ribosomal DNA sequences of coccoid green algae within the Chlorella and the Parachlorella clades of the Chlorellaceae confirmed that strain BAFC CA10 is unequivocally related to the P. kessleri strains. Our results suggest that P. kessleri should be added to the list of species capable of adapting to live in extremely acidic environments.