Iara Rocchetta

Chromium- and copper-induced inhibition of photosynthesis in Euglena gracilis analysed on the single-cell level by fluorescence kinetic microscopy.

Here, we investigated effects of copper (Cu) and chromium (Cr) toxicity on two contrasting strains of Euglena gracilis, with and without chloroplasts, grown in culture media promoting either phototrophic or heterotrophic growth. This led to insights into Cr/Cu toxicity mechanisms and into the regulation of phototrophic vs heterotrophic metabolism. Our data strongly suggest that in Cu(2+) and Cr(6+) stressed Euglena photosynthesis is the primary target of damage. In the applied light conditions, this was mainly damage to the photosystem II reaction centre, as shown by single-cell measurements of photochemical fluorescence quenching. Respiration and photosynthetic dark reactions were less sensitive. The malfunctioning photosynthesis enhanced production of reactive oxygen species (mainly superoxide), leading to elevated amounts of carotenoid degradation products. At higher metal concentrations in chloroplast-containing cells, but not white cells, this oxidative stress resulted in increased respiratory oxygen uptake, likely by damage to mitochondria. During growth in nutrient solution promoting heterotrophic metabolism, the cells were able to repair the metal-induced damage to photosynthesis, moderating the inhibition of photochemistry. Growth in medium forcing the cells into photosynthesis increased the investment in photosynthetic pigments. Comparison of the two Euglena strains surprisingly showed that the previously metal-resistant strain lost this resistance during culture.

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.

Oxidative Stress in Vero Cells Infected with Vesicular Stomatitis Virus

Viral-induced apoptosis might be mediated by oxidative stress. It has already been described that cell death in vesicular stomatitis virus (VSV)-infected cells occurs by apoptosis. In this study, oxidative stress parameters present in VSV-infected Vero cells were analyzed. Lipid peroxides (LP) were evaluated in cellular extracts and expressed as thiobarbituric acid-reactive substances. LP levels exhibited a rise at different times post infection, according to the multiplicity of infection (MOI), while the presence of cycloheximide determined a reduction on LP. Also, an increase in protein degradation products and a decrease in polyunsaturated fatty acids content was observed, indicating that cellular proteins and lipids began to be susceptible to degradation during VSV infection. In addition, we analyzed cell viability of VSV-infected Vero cells, which were incubated in the presence of butylated hydroxyanisole. This antioxidant was able to protect Vero cells, at least at MOIs assayed in this study, and to reduce viral yield only when VSV infection was done at MOI 0.05. Further, superoxide dismutases, which occupy the first step within the antioxidant enzyme cascade, also exhibit a rise in VSV-infected Vero cells, at different MOI. These results suggest that both an oxidative stress and an antioxidative cell response precede the induction of apoptosis by VSV.

Chromium induced stress conditions in heterotrophic and auxotrophic strains of Euglena gracilis.

Oxidative stress parameter and antioxidant defense compound as well as enzyme activity were studied in relation to different Cr(VI) concentrations (0, 10, 20, 40 μM) in two strains of Euglena gracilis, one isolated from a polluted river (MAT) and the other acquired from a culture collection (UTEX). Chromium toxicity was measured in the auxotrophic and obligated heterotrophic variants of the two strains. Chromium uptake was higher in auxotrophic cultures, reflected by their higher cell proliferation inhibition and lower IC50 levels compared to heterotrophic ones. In the Cr(VI) treatments a reduction of chlorophyll a and b ratio (Chl a/Chl b) was observed, the ratio of protein to paramylon content was augmented, and total lipid content increased, having the auxotrophic strains the highest values. TBARS content increased significantly only at 40 μM Cr(VI) treatment. Unsaturated fatty acids also increased in the Cr(VI) treatments, with the higher storage lipid (saturated acids) content in the heterotrophic cells. The antioxidant response, such as SOD activity and GSH content, increased with chromium concentration, showing the highest GSH values in the heterotrophic cultures and the SOD enzyme participation in chromium toxicity. The MAT strain had higher IC50 values, higher carbohydrate and saturated acid content, and better response of the antioxidant system than the UTEX one. This strain isolated from the polluted place also showed higher GSH content and SOD activity in control cells and in almost all treated cultures. SOD activity reached a 9-fold increase in both MAT strains. These results suggest that tolerance of MAT strain against Cr(VI) stress is not only related to GSH level and/or biosynthesis capacity but is also related to the participation of the SOD antioxidant enzyme.

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.