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Optimization of the C11-BODIPY581/591 Dye for the Determination of Lipid Oxidation in Chlamydomonas reinhardtii by Flow Cytometry
Abstract
Lipid oxidation is a recognized end point for the study of oxidative stress and is an important parameter to describe the mode of micropollutant action on aquatic microorganisms. Therefore, the development of quick and reliable methodologies probing the oxidative stress and damage in living cells is highly sought. In the present proof-of-concept work, we examined the potential of the fluorescent dye C11-BODIPY(591/581) to probe lipid oxidation in the green microalga Chlamydomonas reinhardtii. C11-BODIPY(591/581) staining was combined with flow cytometry measurements to obtain multiparameter information on cellular features and oxidative stress damage within single cells. First, staining conditions were optimized by exploring the capability of the dye to stain algal cells under increasing cell and dye concentrations and different staining procedures. Then lipid oxidation in algae induced by short- and long-term exposures to the three metallic micropollutants, copper, mercury, and nanoparticulate copper oxide, and the two organic contaminants, diethyldithiocarbamate (DDC) and diuron was determined. In this work we pointed out C11-BODIPY(591/581) applicability in a wide range of exposure conditions, including studies of oxidation as a function of time and that it is suitable for in vivo measurements of lipid oxidation due to its high permeation and stability in cells and its low interference with algal autofluorescence. © 2013 International Society for Advancement of Cytometry.
... Algae were grown under axenic conditions in 4 × diluted Tris-acetate-phosphate medium (Harris, 2009) at 20 • C and under continuous agitation of 100 rpm in a specialized incubator (Infors, Bottmingen, Switzerland) and an illumination of 110 mol phot m −2 s −1 . At the mid-exponential growth phase, attained after 62-66 h, C. reinhardtii was centrifuged at 2083 × g for 5 min and resuspended in experimental medium containing 10 −3 M 2-(N-morpholino)ethanesulfonic acid (MES, Sigma-Alrdich, Buchs, Switzerland) at pH 6.8 enriched with the macronutrients in the same concentration as the growth medium (Cheloni and Slaveykova, 2013) and Cu added as CuSO 4 (Sigma Aldrich, Buchs, Switzerland). The algal density in the exposure medium was kept at 10 6 cells mL −1 . ...
... For each run the acquisition threshold was set at 20,000 events on the forward scatter (FSC-H) using a flow rate of 14 L min −1 . The algal cells were discriminated from other events adopting the gating strategy as previously described (Cheloni and Slaveykova, 2013). Absolute cell counting was achieved by dividing the number of events present in the gate corresponding to the algal cells, by the volume of the analyzed sample. ...
... Lipid oxidation and membrane permeability alteration were investigated by C11-BODIPY 581/591 (Life Technologies Europe B.V., Zug, Switzerland) and propidium iodide (PI) (Sigma-Aldrich, Buchs, Switzerland), respectively. The staining procedures were optimized for each dye. 5 M CellROX green and 7 M PI were employed for 30 min incubation with no intermediate washing steps, while C11-BODIPY 581/591 staining was performed following the formerly optimized procedure (Cheloni and Slaveykova, 2013). To discriminate between positively stained cells from negative control an equivalent gating strategy was applied for CellROX green and C11-BODIPY 581/591 (Cheloni and Slaveykova, 2013). ...
... To explore the potential of different contaminants to induce cellular oxidative stress, C. reinhardtii was exposed for 2 h to: 10 −9 M and 10 −7 M MeHg, 10 −8 M and 10 −7 M Hg, 10 −5 M, 5 × 10 −6 M and 10 −6 M Cu, 50 and 10 mg· L −1 CuO-NPs and 10 −7 M and 10 −6 M diuron. These concentrations of Cu, Hg, CuO-NPs and diuron were chosen based on the previous results for lipid peroxidation in C. reinhardtii obtained by FCM [40]. The algal suspensions were exposed to each contaminant for 30, 60, 90 and 120 min. ...
... To explore the capabilities of the 2D-assembly to determine the contaminant induced oxidative stress, the cells of C. reinhardtii where exposed to increasing Cu concentrations, stained with CellROX ® Green and observed with fluorescence microscopy. Cu was chosen since it is known to induce oxidative stress in algae [2,40]. Indeed, high concentrations of dissolved Cu induced ROS accumulation and oxidative stress in C. reinhardtii [6,40], Pseudokirchneriella subcapitata and Chlorella vulgaris [51]. ...
... Cu was chosen since it is known to induce oxidative stress in algae [2,40]. Indeed, high concentrations of dissolved Cu induced ROS accumulation and oxidative stress in C. reinhardtii [6,40], Pseudokirchneriella subcapitata and Chlorella vulgaris [51]. ...
An alternative current (AC) dielectrophoretic lab-on-chip setup was evaluated as a rapid tool of capture and assembly of microalga Chlamydomonas reinhardtii in two-dimensional (2D) close-packed arrays. An electric field of 100 V·cm-1, 100 Hz applied for 30 min was found optimal to collect and assemble the algae into single-layer structures of closely packed cells without inducing cellular oxidative stress. Combined with oxidative stress specific staining and fluorescence microscopy detection, the capability of using the 2D whole-cell assembly on-chip to follow the reactive oxygen species (ROS) production and oxidative stress during short-term exposure to several environmental contaminants, including mercury, methylmercury, copper, copper oxide nanoparticles (CuO-NPs), and diuron was explored. The results showed significant increase of the cellular ROS when C. reinhardtii was exposed to high concentrations of methylmercury, CuO-NPs, and 10-5 M Cu. Overall, this study demonstrates the potential of combining AC-dielectrophoretically assembled two-dimensional algal structures with cell metabolic analysis using fluorescence staining, as a rapid analytical tool for probing the effect of contaminants in highly impacted environment.
... Unfortunately, the mechanism by which oxidation acts on BP dye remains unclear. There is abundant evidence that BP oxidation is not caused by the initiators of the lipid peroxidation but rather by a variety of oxy-radicals and peroxynitrate [38,41], and that different products may be formed depending on the oxidant used [42]. Another controversy relates to C11-BODIPY (581/591) sensitivity to oxidation. ...
... It has been demonstrated that conjugated diene connecting the phenyl moiety with the BODIPY core is a target for free radicalmediated oxidation [42]. However, data on the response of BODIPY to different oxidative systems within living cells are contradictory [38,[40][41][42]71]. Here, we tested various protocols to establish a positive control for BODIPY-C11 oxidation. ...
... This matches the results obtained by Sakharov et al. [71] in which C11-BODIPY oxidation was barely visible in cells treated with 5 mM H 2 O 2 , washed and then stained. In another study the percentage of the oxidized algae cells exposed for 30 min to a mixture of BODIPY and 5 mM H 2 O 2 was comparable to those obtained in a treatment in which the cells were exposed to H 2 O 2 , washed and then stained [41]. The lack of BP oxidation in our experiment can be explained by the application of different oxidizer with much smaller, non-lethal doses (data not shown) that were not sufficient for a prominent endogenous ROS generation or were immediately countered by antioxidant systems within CHO-K1 cells. ...
The permeabilized condition of the cell membrane after electroporation can last minutes but the underlying mechanisms remain elusive. Previous studies suggest that lipid peroxidation could be responsible for the lasting leaky state of the membrane. The present study aims to link oxidation within the plasma membrane of live cells to permeabilization by electric pulses. We have introduced a method for the detection of oxidation by ratiometric fluorescence measurements of BODIPY-C11 dye using total internal reflection fluorescence (TIRF) microscopy, limiting the signal to the cell membrane. CHO-K1 cells were cultured on glass coverslips coated with an electroconductive indium tin oxide (ITO) layer, which enabled electroporation with micro- and submicrosecond pulses. No oxidation was observed with the electric field directed towards the ITO (cathode), even at field strengths much higher than that needed for permeabilization. Oxidation was readily detectable with the opposite polarity of pulses, but with the threshold higher than the permeabilization threshold. Moreover, a decrease in the medium conductance had opposite effects on permeabilization and lipid oxidation (it enhanced the former and suppressed the latter). We conclude that lipid oxidation can indeed occur at the plasma membrane after electric pulses, but it is not the cause of lasting membrane permeabilization.
... Usually LPO is quantified by measuring by-products such as malondialdehyde (MDA) and other carbonyls. Recently, a method using a lipophilic florescent dye C11-BODIPY 581/591 (4,4-difluoro-5-(4-phenyl-1,3-butadienyl)-4-bora-3a,4adiaza-s-indacene-3-undecanoid acid) has been successfully used to measure LPO in C. reinhardtii exposed to different stressors (Cheloni and Slaveykova, 2013;Melegari et al., 2013). The direct and indirect formation of ROS in photosynthetic organisms can damage or modify essential proteins and lipid components present in the thylakoid membrane of chloroplasts, leading to a decrease in photosynthetic performance (Asada, 2006;Sharma et al., 2012). ...
... The probe C 11 -BODIPY 581/591 was used for measuring LPO in exposed algal cells (adapted from Cheloni and Slaveykova, 2013). This probe is a fatty acid analogue with specific fluorescence properties, which can easily enter the lipid bilayer and be subject to oxidation by oxyl-radicals together with the endogenous fatty acids, once inside the cellular membrane (Cheloni and Slaveykova, 2013). ...
... The probe C 11 -BODIPY 581/591 was used for measuring LPO in exposed algal cells (adapted from Cheloni and Slaveykova, 2013). This probe is a fatty acid analogue with specific fluorescence properties, which can easily enter the lipid bilayer and be subject to oxidation by oxyl-radicals together with the endogenous fatty acids, once inside the cellular membrane (Cheloni and Slaveykova, 2013). Stock solutions of 2.5 mM were prepared in DMSO and stored in aliquots at −20°C until use. 100 μl of assay working solutions were prepared by diluting the probe in the assay buffer (final concentration 5 μM) with the test compounds. ...
The toxicity of biocides can be associated with the formation of reactive oxygen species (ROS) and subsequent oxidative damage, interfering with the normal function of photosynthetic organisms. This study investigated the formation and effects of ROS in the unicellular green algae Chlamydomonas reinhardtii exposed to three environmentally relevant biocides, aclonifen, dichlofluanid and triclosan. After a first screening to identify which biocides induced ROS, a 24 h multi-endpoint analysis was used to verify the possible consequences. A battery of high-throughput methods was applied in algae for measuring ROS formation, reduced glutathione (GSH), lipid peroxidation (LPO), photosystem (PS) II performance and pigments (chlorophylls a, b and carotenoids).
... Analyses were conducted in triplicate with independent batches of algae and exposure media that were prepared on different days.For each acquisition, a threshold of 10,000 events in the gate corresponding to algal cells was selected. Details on the flow cytometry gating strategy can be found in the Supplemental information (Figure S1andFigure S2), however, it mainly followed procedures described inCheloni et al. (2013Cheloni et al. ( , 2014.28,29 The proportions of cells that were present in the different gates of interest were retrieved with the CFlow Plus program, in addition to the average values of the different parameters of interest.Statistical analysesStatistical analyses were performed in SigmaPlot (v12.0). ...
... Analyses were conducted in triplicate with independent batches of algae and exposure media that were prepared on different days.For each acquisition, a threshold of 10,000 events in the gate corresponding to algal cells was selected. Details on the flow cytometry gating strategy can be found in the Supplemental information (Figure S1andFigure S2), however, it mainly followed procedures described inCheloni et al. (2013Cheloni et al. ( , 2014.28,29 The proportions of cells that were present in the different gates of interest were retrieved with the CFlow Plus program, in addition to the average values of the different parameters of interest.Statistical analysesStatistical analyses were performed in SigmaPlot (v12.0). ...
Cerium (Ce) is a rare earth element that is incorporated in numerous consumer products, either in its cationic form or as engineered nanoparticles (ENPs). Given the propensity of small oxide particles to dissolve, it is unclear if biological responses induced by ENPs will be due to the nanoparticles themselves or rather to their dissolution. This study provides the foundation for the development of transcriptomic biomarkers that are specific for ionic Ce in the freshwater alga, Chlamydomonas reinhardtii. exposed either to ionic Ce or to two different types of small Ce ENPs (uncoated, ∼10 nm or citrate coated, ∼ 4 nm). Quantitative reverse transcription PCR was used to analyse mRNA levels of four ionic Ce specific genes (Cre17g.737300, MMP6, GTR12 and HSP22E) that were previously identified by whole transcriptome analysis in addition to two oxidative stress biomarkers (APX1 and GPX5). Expression was characterized for exposures to 0.03 to 3 µM Ce, for 60 to 360 minutes and for pH 5.0-8.0. Near linear concentration-response curves were obtained for the ionic Ce and as a function of exposure time. Some variability in the transcriptomic response was observed as a function of pH, which was attributed to the formation of metastable Ce species in solution. Oxidative stress biomarkers analysed at transcriptomic and cellular levels confirmed that different effects were induced for dissolved Ce in comparison to Ce ENPs. The measured expression levels confirmed that changes in Ce speciation and the dissolution of Ce ENPs greatly influence Ce bioavailability.
... The probe C 11 -BODIPY 581/591 (adapted from Cheloni and Slaveykova, 2013) was used to determine LPO after 24 h exposure to bifenox and metribuzin (Almeida et al., 2017). This is a fatty acid analogue probe with specific fluorescence properties that can be oxidized by oxyl-radicals together with the endogenous fatty acids (Cheloni and Slaveykova, 2013). Stock solutions of 2.5 mM were prepared in anhydrous DMSO and stored in aliquots at −20°C until use. ...
... However, a depletion in GSH levels at concentrations higher than 1 × 10 3 nM was observed, which resulted in an exponential increase in ROS formation. This decrease in GSH levels and increase in ROS resulted in a significant increase in LPO levels, indicating the increased production of oxyl-radicals such as HO % , ROO % , RO % and ONOO-(indicated by the fluorescent dye C 11 -BODIPY 581/591 ; Cheloni and Slaveykova, 2013). LPO levels decreased again to basal levels at the three highest tested concentrations (from 1 × 10 4 nM), even though the levels of ROS continued to increase, showing that other scavenging processes may have responded to the increase of radical species. ...
The widespread presence of herbicides in the aquatic environment has raised awareness about the need to develop further in depth ecotoxicological risk assessments, more specifically on potential effects on photosynthetic organisms as microalgae. The majority of the information available regarding the toxicity of herbicides towards microalgae is related to traditional toxicological and regulatory-relevant endpoints such as growth inhibition, leaving a significant gap on knowledge regarding underlying interactions and damage to biological targets. In this context, this study aimed to supplement the general toxicity information of bifenox and metribuzin in the microalgae Chlamydomonas reinhardtii using a battery of selected high-throughput methods. This multiple-endpoint approach included the measurement of formation of reactive oxygen species (ROS), alterations in reduced glutathione (GSH) content, formation of lipid peroxidation (LPO), photosystem II (PSII) performance and loss of photosynthetic pigments after 24 h exposure. Results obtained showed that both herbicides caused a concentration-dependent increase in ROS formation, with bifenox showing higher but less reactive ROS. This increase in ROS production by bifenox and metribuzin was followed by alterations in the antioxidant capacity of algae, oxidative damage in the form of LPO and alterations in pigment content. Furthermore, both herbicides impacted the photosynthetic activity of algae, as seen by alterations in the maximum and effective quantum efficiency of PSII, PSII photochemistry and energy dissipation pathways, impact in the water-splitting apparatus and reduction in the electron transport rate. The inhibitory effect of metribuzin on photosynthetic processes/components was larger than that seen for bifenox. The impact of bifenox and metribuzin in the photosynthetic processes of C. reinhardtii seems to be in close association with the formation of ROS and consequent oxidative stress and damage in algal cells. Overall, this study showed that the high-throughput methods developed could successfully characterise both potential Modes of Action and adverse effects of bifenox and metribuzin in C. reinhardtii.
... The oxyl and peroxyl radicals associated with lipid peroxidation (LPO) was measured in L. minor fronds by the fluorescent dye C11-BODIPY 581/591 (Invitrogen Molecular Probe, Eugene, Oregon, USA) as originally described by Cheloni and Slaveykova (2013) with minor modifications by Almeida et al. (2017). BODIPY is a fatty acid analogue with specific fluorescence properties which can be subject to oxidation by oxyl-radicals together with endogenous fatty acids inside the cellular membrane and thus acts as an indicator of potential LPO (Cheloni and Slaveykova, 2013). ...
... The oxyl and peroxyl radicals associated with lipid peroxidation (LPO) was measured in L. minor fronds by the fluorescent dye C11-BODIPY 581/591 (Invitrogen Molecular Probe, Eugene, Oregon, USA) as originally described by Cheloni and Slaveykova (2013) with minor modifications by Almeida et al. (2017). BODIPY is a fatty acid analogue with specific fluorescence properties which can be subject to oxidation by oxyl-radicals together with endogenous fatty acids inside the cellular membrane and thus acts as an indicator of potential LPO (Cheloni and Slaveykova, 2013). Stock solutions of 5 mM were prepared in DMSO and stored at −20°C until use. ...
High dose rates of ionizing radiation have been reported to cause adverse effects such as reduction in reproduction and growth, and damage to protein and lipids in primary producers. However, the relevant effects of ionizing radiation are still poorly understood in aquatic plants. This study was intended to characterize the biological effects and modes of action (MoAs) of ionizing radiation using gamma radiation as the prototypical stressor and duckweed Lemna minor as a model organism. Lemna minor was exposed to 1, 14, 24, 46, 70 mGy/h gamma radiation dose rates from a cobalt-60 source for 7 days following the testing principles of the OECD test guideline 221. A suite of bioassays was applied to assess the biological effects of gamma radiation at multiple levels of biological organization, including detection of reactive oxygen species (ROS), oxidative stress responses (total glutathione, tGSH; lipid peroxidation, LPO), DNA damage, mitochondrial dysfunctions (mitochondrial membrane potential, MMP), photosynthetic parameters (chlorophyll a, chl a; chlorophyll b, chl b; carotenoids; Photosystem II (PSII) performance; CO2 uptake), intercellular signaling (Ca2+ release) and growth. Gamma radiation increased DNA damage, tGSH level and Ca2+ content together with reduction in chlorophyll content, maximal PSII efficiency and CO2 uptake at dose rates between 1 and 14 mGy/h, whereas increases in cellular ROS and LPO, inhibition of MMP and growth were observed at higher dose rates (≥24 mGy/h). A network of toxicity pathways was proposed to portray the causal relationships between gamma radiation-induced physiological responses and adverse outcomes to support the development of Adverse Outcome Pathways (AOPs) for ionizing radiation-mediated effects in primary producers.
... Additionally fluorescent probes were employed to detect whether roots colonized by PcO6 had altered ROS production when grown with exposure to CuO NPs. CellROX Deep Red was used to examine the intracellular accumulation of ROS [26], fluorescence from C11-BODIPY 581-591 was imaged to determine the production of lipid peroxides [27] and the accumulation of superoxide anion was studied with fluorescence from dihydroethidium (DHE) [28]. Another dye, SYTOX Blue, was used to determine how treatments modified cell death at the surface of the root tips [29]. ...
... The accumulation of lipid peroxides was detected with C-11 BODIPY 581/591 (Life Technologies, Carlsbad CA) [26,27]. Root tips were immersed in 10 μM dye prepared by dilution of the DMSO stock solution in sterile distilled water for 30 min and briefly rinsed with distilled water. ...
Nanoparticle (NPs) containing essential metals are being considered in formulations of fertilizers to boost plant nutrition in soils with low metal bioavailability. This paper addresses whether colonization of wheat roots by the bacterium, Pseudomonas chlororaphis O6 (PcO6), protected roots from the reduced elongation caused by CuO NPs. There was a trend for slightly elongated roots when seedlings with roots colonized by PcO6 were grown with CuO NPs; the density of bacterial cells on the root surface was not altered by the NPs. Accumulations of reactive oxygen species in the plant root cells caused by CuO NPs were little affected by root colonization. However, bacterial colonization did reduce the extent of expression of an array of genes associated with plant responses to stress induced by root exposure to CuO NPs. PcO6 colonization also reduced the levels of two important chelators of Cu ions, citric and malic acids, in the rhizosphere solution; presumably because these acids were used as nutrients for bacterial growth. There was a trend for lower levels of soluble Cu in the rhizosphere solution and reduced Cu loads in the true leaves with PcO6 colonization. These studies indicate that root colonization by bacterial cells modulates plant responses to contact with CuO NPs.
... Upon oxidation the BODIPY™ 581/591 C11 exhibit a shift of the fluorescence emission peak from ~590 nm to ~510 nm, resulting in decrease of red fluorescence [36,37]. Thus, the intensity of red fluorescence can be used as an indicator of lipid peroxidation under oxidative stress [38,39]. We performed a lipid peroxidation assay in accordance with the manufacturer's instructions and previously published protocols [38,39]. ...
... Thus, the intensity of red fluorescence can be used as an indicator of lipid peroxidation under oxidative stress [38,39]. We performed a lipid peroxidation assay in accordance with the manufacturer's instructions and previously published protocols [38,39]. Briefly, cells were seeded onto 96-well clean bottom plates at the density of 5000 cells per well and treated with different concentration of nanoparticles for 24 h. ...
Iron oxide-based nanoparticles have been repeatedly shown to affect lysosomal-mediated signaling. Recently, nanoparticles have demonstrated an ability to modulate autophagic flux via lysosome-dependent signaling. However, the precise underlying mechanisms of such modulation as well as the impact of cellular genetic background remain enigmatic. In this study, we investigated how lysosomal-mediated signaling is affected by iron oxide nanoparticle uptake in three distinct hepatic cell lines. We found that nanoparticle-induced lysosomal dysfunction alters sub-cellular localization of pmTOR and p53 proteins. Our data indicate that alterations in the sub-cellular localization of p53 protein induced by nanoparticle greatly affect the autophagic flux. We found that cells with high levels of Bcl-2 are insensitive to autophagy initiated by nanoparticles. Altogether, our data identify lysosomes as a central hub that control nanoparticle-mediated responses in hepatic cells. Our results provide an important fundamental background for the future development of targeted nanoparticle-based therapies.
... Two independent assays were used to monitor lipid peroxidation. First, we used Bodipy 581/591-C11, which is a fluorescent probe mimicking unsaturated fatty acids 26 . We also investigated formation of the highly specific lipid peroxidation product 8-isoprostane with an enzyme immunoassay (EIA) 27 . ...
... Subjects. Samples were collected from 8 healthy male volunteers (age range [19][20][21][22][23][24][25][26][27] participating in a randomized controlled human endotoxemia study 45 . This study was approved by the local ethics committee of the Radboud University Nijmegen Medical Centre (CMO-number 2012/455). ...
Ethylene is a major plant hormone mediating developmental processes and stress responses to stimuli such as infection. We show here that ethylene is also produced during systemic inflammation in humans and is released in exhaled breath. Traces of ethylene were detected by laser spectroscopy both in vitro in isolated blood leukocytes exposed to bacterial lipopolysaccharide (LPS) as well as in vivo following LPS administration in healthy volunteers. Exposure to LPS triggers formation of ethylene as a product of lipid peroxidation induced by the respiratory burst. In humans, ethylene was detected prior to the increase of blood levels of inflammatory cytokines and stress-related hormones. Our results highlight that ethylene release is an early and integral component of in vivo lipid peroxidation with important clinical implications as a breath biomarker of bacterial infection.
... Determination of lipid peroxidation The lipid peroxidation level of the algal cells was determined at 48 and 96 h using the cell permeable indicator C11-BODIPY 581/591 (Life Technologies, USA) (Cheloni and Slaveykova 2013). The microalgae culture (150 μL) was withdrawn into black 96well microplates. ...
There has been concern over the adverse ecotoxicological effects of atrazine and endosulfan on microalgae. This study aimed to assess the effects of these two widely used pesticides on growth, pigmentation, and oxidative response of microalgal isolates from a farmland and a eutrophic lake in Malaysia, in comparison with the model species Pseudokirchneriella subcapitata. Results showed that the microalgae originated from the eutrophic lake were generally more sensitive to the pesticides than those from the farmland. The microalgae were more sensitive to atrazine (EC50 = 43.07–> 5000 μg L⁻¹) than endosulfan (EC50 = 1.51–> 50 mg L⁻¹). Amongst the microalgae, Scenedesmus arcuatus was most sensitive to atrazine (EC50 = 43.07 μg L⁻¹) while Chlorella sp. 1 was most sensitive to endosulfan (EC50 = 1.51 mg L⁻¹). Microalgae from the farmland were generally very tolerant to endosulfan (EC50 > 50 mg L⁻¹). Photosynthetic pigment content (pg cell⁻¹) increased in S. arcuatus after exposure to atrazine while the content decreased in most of the microalgae after exposure to endosulfan. Oxidative response to the pesticides varied amongst the tested microalgae and time point measured. Both ROS levels and lipid peroxidation decreased in Chlorella sp. 5 after exposure to atrazine at 96 h compared to 48 h. In S. arcuatus, there was no pronounced increase in SOD and catalase activities despite the increase in ROS and lipid peroxidation after exposure to atrazine. Indigenous microalgae such as S. arcuatus could be a useful bioassay organism for toxicity testing of the pesticides while tolerant species from the farmland could be useful for bioremediation of endosulfan contamination.
... As mentioned above, ROS are involved in oxidative damage to cell membrane constituents. Hence, complementary analysis of lipid peroxidation (Cheloni and Slaveykova, 2013;Melegari et al., 2013) might make it possible to link the effects observed on the cytoplasmic membrane potential with those observed on intracellular ROS content. ...
This study screened binary mixtures of pesticides for potential synergistic interaction effects on growth of the marine microalgae Tisochrysis lutea and Skeletonema marinoi. It also examined the single and combined effects of three of the most toxic substances on microalgal physiology. Single substances were first tested on each microalgal species to determine their respective EC 50 and concentration-response relationships. The toxicity of six and seven binary mixtures was then evaluated in microplate experiments on the growth of T. lutea and S. marinoi, respectively, using two mixture modelling approaches: isobolograms and the MIXTOX tool, based on Concentration Addition (CA) or Independent Action (IA) models. Significant cases of antagonism (for both species) and synergism (for S. marinoi) were observed for the mixtures of isoproturon and spiroxamine, and isoproturon and metazachlor, respectively. These two mixtures, together with that of isoproturon and diuron, for which additivity was observed, were further studied for their impacts on the physiology of each species. Exposures were thus made in culture flasks at three concentrations, or concentration combinations for mixtures, selected to cause 25%, 50% and 75% growth rate inhibition. The effects of the selected pesticides singly and in combination were evaluated at three perceived effect concentrations on esterase metabolic activity, relative lipid content, cytoplasmic membrane potential and reactive oxygen species (ROS) content by flow cytometry, and on photosynthetic quantum yield (ϕ’ M ) by PAM-fluorescence. Isoproturon and diuron singly and in mixtures induced 20–40% decreases in ϕ’ M which was in turn responsible for a significant decrease in relative lipid content for both species. Spiroxamine and metazachlor were individually responsible for an increase in relative lipid content (up to nearly 300% for metazachlor on S. marinoi), as well as cell depolarization and increased ROS content. The mixture of isoproturon and metazachlor tested on S. marinoi caused a 28–34% decrease in ϕ’ M that was significantly higher than levels induced by each of substances when tested alone. This strong decrease in ϕ’ M could be due to a combined effect of these substances on the photosynthetic apparatus, which is likely the cause of the synergy found for this mixture.
... Oxidation of the polyunsaturated butadienyl portion of the C11-BODIPY resulted in a shift of the fluorescence emission peak from ~ 590 to ~ 510 nm. The relative levels of lipid peroxidation and Fe 2+ were measured by flow cytometry, which was quantified by the FITC/PE value and the PE value, respectively (Cheloni and Slaveykova 2013;Mei et al. 2020). ...
Epidermal stem cells (ESCs) are critical for skin regeneration and repair. Previous studies have shown that ESCs are susceptible to oxidative stress, which in turn leads to lipid peroxidation and affects skin repair. Our study aims to explore how ESCs resist lipid peroxidation. By performing proteomics analysis, we found that the expression of Acyl-CoA thioesterase 7 (ACOT7) was positively correlated with the concentration of transferrin. Overexpression adenovirus vectors of ACOT7 were constructed and transfected into ESCs. Levels of lipid peroxidation by flow cytometry, cell viabilities, and MDA levels were measured. The results revealed that ACOT7 could inhibit lipid peroxidation, reduce the level of malondialdehyde (MDA), and improve the survival rate of ESCs induced by H 2 O 2 , Erastin, and RSL3. Our data suggest that ACOT7 has an effect on protecting ESCs against iron-dependent lipid peroxidation.
... This reaction is commonly named lipid peroxidation [96]. In order to quantify this membrane damage, the dye BODIPY 581/591 C11 (Thermo Fisher Scientific, MA, USA) is used [97]. This dye is a fatty acid analogue with particular fluorescent properties. ...
Suicidal erythrocyte death or eryptosis contributes to or even accounts for anemia in a wide variety of clinical conditions, such as iron deficiency, dehydration, hyperphosphatemia, vitamin D excess, chronic kidney disease (CKD), hemolytic-uremic syndrome, diabetes, hepatic failure, malignancy, arteriitis, sepsis, fever, malaria, sickle-cell disease, beta-thalassemia, Hb-C and G6PD-deficiency, Wilsons disease, as well as advanced age. Moreover, eryptosis is triggered by a myriad of xenobiotics and endogenous substances including cytotoxic drugs and uremic toxins. Eryptosis is characterized by cell membrane scrambling with phosphatidylserine exposure to the erythrocyte surface. Triggers of eryptosis include oxidative stress, hyperosmotic shock, and energy depletion. Signalling involved in the regulation of eryptosis includes Ca2+ entry, ceramide, caspases, calpain, p38 kinase, protein kinase C, Janus-activated kinase 3, casein kinase 1α, cyclin-dependent kinase 4, AMP-activated kinase, p21-activated kinase 2, cGMP-dependent protein kinase, mitogen- and stress-activated kinase MSK1/2, and ill-defined tyrosine kinases. Inhibitors of eryptosis may prevent anaemia in clinical conditions associated with enhanced eryptosis and stimulators of eryptosis may favourably influence the clinical course of malaria. Additional experimentation is required to uncover further clinical conditions with enhanced eryptosis, as well as further signalling pathways, further stimulators, and further inhibitors of eryptosis. Thus, a detailed description of the methods employed in the analysis of eryptosis may help those, who enter this exciting research area. The present synopsis describes the experimental procedures required for the analysis of phosphatidylserine exposure at the cell surface with annexin-V, cell volume with forward scatter, cytosolic Ca2+ activity ([Ca2+]i) with Fluo3, oxidative stress with 2′,7′-dichlorodihydrofuorescein diacetate (DCFDA), glutathione (GSH) with mercury orange 1(4-chloromercuryphenyl-azo-2-naphthol), lipid peroxidation with BODIPY 581/591 C11 fluorescence, and ceramide abundance with specific antibodies. The contribution of kinases and caspases is defined with the use of the respective inhibitors. It is hoped that the present detailed description of materials and methods required for the analysis of eryptosis encourages further scientists to enter this highly relevant research area.
... T. thermophile regulates its membrane's lipid composition to more rigid configuration by lowering its unsaturated fatty acids and increasing saturated fatty acids. Studies also evidence the lipid peroxidation in rainbow and C. reinhardtii when exposed to CuO NPs [58,59]. The studies also report cytotoxicity of CuO NPs in primary liver cells of cat fish and HepG2 due to generation of ROS [60,61]. ...
Copper oxide nanoparticles (CuO NPs) use has exponentially increased in various applications (such as industrial catalyst, gas sensors, electronic materials, biomedicines, environmental remediation) due to their flexible properties ie large surface area to volume ratio. These broad applications, however, have increased human exposure and thus the potential risk related to their short-and long-term toxicity. Their release in environment has drawn considerable attention which has become an eminent area of research and development. To understand the toxicological impact of CuO NPs, this review summarizes the in vitro and in vivo toxicity of CuO NPs subjected to species (bacterial, algae, fish, rats, human cell lines) used for toxicological hazard assessment. The key factors that influence the toxicity of copper oxide nanoparticles such as particle shape, size, surface functionalization, time-dose interaction .....IEt
... Synergistic interactions between the two stressors with higher OS in combined than in individual treatments were found (Cheloni et al. 2016). The above examples illustrated that the potential of CuO-NPs to induce OS was also modulated by the test medium composition (von Moos et al. 2015), as well as the interactions with other varying environmental factors, including solar radiation (Cheloni and Slaveykova, 2013;Cheloni et al. 2016). ...
We report a of the symposium “Stress and Protists: No life without stress”, which was held in September 2015 on the VII European Congress of Protistology in partnership with the International Society of Protistologists (Seville, Spain). We present an overview on general comments and concepts on cellular stress which can be also applied to any protist. Generally, various environmental stressors may induce similar cell responses in very different protists. Two main topics are reported in this manuscript: i) metallic nanoparticles as environmental pollutants and stressors for aquatic protists, and ii) ultraviolet radiation–induced stress and photoprotective strategies in ciliates. Model protists such as _Chlamydomonas reinhardtii_ and _Tetrahymena thermophila_ were used to assess stress caused by nanoparticles while stress caused by ultraviolet radiation was tested with free living planktonic ciliates as well as with the symbiont-bearing model ciliate _Paramecium bursaria_. For future studies, we suggest more intensive analyses on protist stress responses to specific environmental abiotic and/or biotic stressors at molecular and genetic levels up to ecological consequences and food web dynamics.
... ROS can cause lipid peroxidation, which can be measured with the Bodipy581/591-C11 probe. Bodipy581/591-C11 contains alkene groups mimicking unsaturated fatty acids and making it susceptible to peroxidation 31 . This probe incorporates into cellular membranes and peroxidation causes a blue-shift of its fluorescence over time, which can be measured by flow cytometry (Fig. 1b). ...
Dendritic cells (DCs) present foreign antigen in major histocompatibility complex (MHC) class I
molecules to cytotoxic T cells in a process called cross-presentation. An important step in this process is
the release of antigen from the lumen of endosomes into the cytosol, but the mechanism of this step is
still unclear. In this study, we show that reactive oxygen species (ROS) produced by the NADPH-oxidase
complex NOX2 cause lipid peroxidation, a membrane disrupting chain-reaction, which in turn results in
antigen leakage from endosomes. Antigen leakage and cross-presentation were inhibited by blocking
ROS production or scavenging radicals and induced when using a ROS-generating photosensitizer.
Endosomal antigen release was impaired in DCs from chronic granulomatous disease (CGD) patients
with dysfunctional NOX2. Thus, NOX2 induces antigen release from endosomes for cross-presentation
by direct oxidation of endosomal lipids. This constitutes a new cellular function for ROS in regulating
immune responses against pathogens and cancer.
... Cell pellets were washed twice with the CuO-NPs free medium and re-suspended in it to a final cell density of 10 6 cells m L −1 . The experimental medium contained 10 −3 M 2-(N-morpholino) ethanesulfonic acid (MES, Sigma-Alrdich, Buchs, Switzerland) at pH 6.8 enriched with the macronutrients in the same concentration as the growth medium (Cheloni and Slaveykova, 2013) and CuO-NPs. ...
The present study explores the effect of light with different spectral composition on the stability of CuO-nanoparticle (CuO-NP) dispersions and their effects to green alga Chlamydomonas reinhardtii. The results showed that simulated natural light (SNL) and light with enhanced UVB radiation (UVR*) do not affect the dissolution of CuO-NPs as compared to light irradiation conditions typically used in laboratory incubator (INC). Comparable values of ζ-potential and hydrodynamic size during 24h were found under all studied conditions. Concentrations of CuO-NPs below 1mgL(-1) do not attenuate the light penetration in the algal suspensions in comparison with NP-free system. Exposure to a combination of 8μgL(-1) or 0.8mgL(-1) CuO-NPs and INC or SNL has no significant effect on the algal growth inhibition, algal fluorescence and membrane integrity under short-term exposure. However, an enhancement of the percentage of cells experiencing oxidative stress was observed upon exposure to 0.8mgL(-1) CuO-NPs and SNL for 4 and 8h. Combination of UVR* and 0.8mgL(-1) CuO-NPs resulted in synergistic effects for all biological endpoints. Despite the photocatalytic properties of CuO-NPs no significant increase in abiotic reactive oxygen species (ROS) production under simulated solar radiation was observed suggesting that the synergistic effect observed might be correlated to other factors than CuO-NP-mediated ROS photoproduction. Tests performed with CuSO4 confirmed the important role of dissolution as toxicity driving force for lower CuO-NP concentration. However, they failed to clarify the contribution of dissolved Cu on the combined effects at 0.8mgL(-1) CuO-NPs. The results point out the necessity of taking into account the possible interactions between ENPs and changing light conditions when evaluating the potential effects of ENPs to phytoplankton in natural waters.
... Before use, the stock dispersion received a 1 min sonication probe treatment at an amplitude of 100% (130 W, 20 kHz) without pulses (VCX 130,Sonics Vibra Cell,Sonic & Materials Inc.,Newtown,USA) and was diluted in the exposure medium. The exposure medium contained 10 À4 M KH 2 PO 4 ; 1.5 Â 0 À4 M K 2 HPO 4 ; 1.7 Â 10 À3 M NH 4 NO 3 ; 8.5 Â 10 À5 M CaCl 2 Á2H 2 O; 10 À4 M MgSO 4 7H 2 O in 10 À3 M 2-(N-morpholino) ethanesulfonic acid (MES, Sigma Aldrich, Buchs Switzerland) at pH 6.8 (Cheloni and Slaveykova, 2013) to a nominal NP concentration of 10 mg L À1 CuO. The evolution of the hydrodynamic size and zeta-potential in CuO-NP dispersions was determined in the exposure medium after 1, 2, 4 and 24 h by measurement with a Malvern Zetasizer Nano-ZS. ...
... The small differences between DHR 123 and the other two probes are related to their specificity. Carboxy-H 2 DFFDA is activated by H 2 O 2 , NO, ONOO − , O 2 and OH (Gunawan et al., 2013), while C 11 -BODIPY 581/591 reacts to oxyl-radicals such as HO•, ROO•, RO• and peroxynitrite responsible for LPO (Cheloni and Slaveykova, 2013). Although carboxy-H 2 DFFDA and DHR 123 have similar structural features, and are oxidized by similar oxidative/radical mechanisms, carboxy-H 2 DFFDA is generally associated with ROS formation in chloroplasts. ...
Microalgal toxicity tests using integrative endpoints as algal growth are regularly required to analyse the toxicity of potentially hazardous substances in the aquatic environment. However, these do not provide mechanistic information on the toxic mode of action by which contaminants may affect algae. Bottled waters can be used as a substitute for culturing media and should not impose any stress to the cultured organisms. However, certain chemical components can interfere with specific cell targets which are not revealed by general toxicity assays. The present study investigated the sensitivity of flow cytometry (FCM) to analyse sub-lethal effects of different bottled waters to the freshwater microalgae Raphidocelis subcapitata. Several endpoints were analysed including growth rate, natural pigments content, cell size, complexity, viability and cycle, Reactive Oxygen Species (ROS) formation, mitochondrial membrane potential and Lipid Peroxidation (LPO). Additionally, photosystem II (PSII) performance was analysed by PAM fluorometry, to provide further information on the absorption, distribution and use of energy in photosynthesis. Results indicated that the most sensitive endpoints were the oxidative stress related endpoints ROS formation and LPO, pigment content, morphological endpoints as cell size, complexity and cycle, with growth rate being one of the least sensitive. Although being essential macronutrients for algal growth, the chemical elements Ca, Na, Mg, and NH4 were identified as being primarily responsible for the observed toxicological effects to exposed algae. The applied methodology proved to be of high throughput, simultaneously assembling information on morphological, biochemical, and physiological status of algal cells. FCM also showed potential to reveal mechanistic information on the toxic mode of action of the bottled waters before any effects on algal growth was observed. The used approach demonstrated its potential for being integrated into future microalgal toxicity bioassays for testing chemicals to improve the hazard information obtained from currently approved internationally accepted test guidelines.
... Effects of CuO NPs on membrane lipids have been also described for unicellular protozoa T. thermophila by Mortimer et al. (2011): in response to exposure to CuO NPs (80 mg/L), protozoa adjusted its membrane fatty acid composition to more rigid by decreasing the amount of unsaturated fatty acids (C18:3 cis-6, 9, 12 and C18:2 cis-9, 12) and increasing the amount of saturated fatty acids (C18:0 and C16:0). Lipid peroxidation caused by CuO NPs has also been demonstrated in tissue and gills of rainbow trout , bacteria E. coli and Bacillus subtilis (Applerot et al., 2012) and green algae C. reinhardtii (Cheloni & Slaveykova, 2013). ...
Abstract Silver, ZnO and CuO nanoparticles (NPs) are increasingly used as biocides. There is however increasing evidence of their threat to "non-target" organisms. In such a context, the understanding of the toxicity mechanisms is crucial for both the design of more efficient nano-antimicrobials, i.e. for "toxic by design" and at the same time for the design of nanomaterials that are biologically and/or environmentally benign throughout their life-cycle (safe by design). This review provides a comprehensive and critical literature overview on Ag, ZnO and CuO NPs' toxicity mechanisms on the basis of various environmentally relevant test species and mammalian cells in vitro. In addition, factors modifying the toxic effect of nanoparticles, e.g. impact of the test media, are discussed. Literature analysis revealed three major phenomena driving the toxicity of these nanoparticles: (i) dissolution of nanoparticles, (ii) organism-dependent cellular uptake of NPs and (iii) induction of oxidative stress and consequent cellular damages. The emerging information on quantitative structure-activity relationship modeling of nanomaterials' toxic effects and the challenges of extrapolation of laboratory results to the environment are also addressed.
... In recent years, the use of fluorescent probes specifically designed to detect oxidative stress in living cells, probes based on dihydrofluorescein diacetate, has been applied in vitro. A lipophilic fluorescent dye 4,4-difluoro-5-(4-phenyl-1,3-butadienyl)-4bora-3a,4a-diaza-s-indacene-3-undecanoic acid (C11-BOD-IPY 581/591) to probe oxyl-radical-induced lipid oxidation by flow cytometry (FCM) has higher sensitivity and specificity for lipid peroxidation in vitro [50]. ...
Reactive oxygen species- (ROS-) induced lipid peroxidation plays a critical role in cell death including apoptosis, autophagy, and ferroptosis. This fundamental and conserved mechanism is based on an excess of ROS which attacks biomembranes, propagates lipid peroxidation chain reactions, and subsequently induces different types of cell death. A highly evolved sophisticated antioxidant system exists that acts to protect the cells from oxidative damage. In this review, we discussed how ROS propagate lipid peroxidation chain reactions and how the products of lipid peroxidation initiate apoptosis and autophagy in current models. We also discussed the mechanism of lipid peroxidation during ferroptosis, and we summarized lipid peroxidation in pathological conditions of critical illness. We aim to bring a more global and integrative sight to know how different ROS-induced lipid peroxidation occurs among apoptosis, autophagy, and ferroptosis.
... Thus, we next verified that ROS produced by NVs cp could trigger lipid peroxidation. Lipid peroxidation in bone marrow dendritic cells (BMDCs) and bone marrow-derived macrophages (BMDMs) are measured using BODIPY™ 581/591-C11 fluorescence, which displays a shift of fluorescence emission peaks from ~590 nm (red) to ~510 nm (green) upon oxidation by lipid hydroperoxides [33]. Culturing BMDCs and BMDMs in the presence of NVs cp versus NVs resulted in an ~50% increase in BODIPY™ 581/591-C11 fluorescence ( Fig. 2D and Figure S9, Supporting Information). ...
Current efforts to develop novel vaccine nanotechnologies to increase cytotoxic T lymphocytes have met the challenges of the limited efficacy of antigen cross-presentation. Recent studies have uncovered a unique biological mechanism by which activation of the NADPH oxidase 2 (NOX2) complex, a major source of reactive oxygen species (ROS), enhances the cross-presentation by antigen-presenting cells (APCs). Inspired by the NOX2 mechanism, we devise biomineralized nanovaccines named NVscp, which are developed by in situ growth of calcium peroxide on nanovaccines self-assembled with the model antigen ovalbumin. The ∼80 nm NVscp efficiently flow to the draining lymph nodes, where they accumulate within APC endo-/lysosomes, and generate a rapid burst of ROS in response to the acidic endo-/lysosomal environment with the subsequent endo-/lysosomal lipid peroxidation. Accompanied by the process, NVscp stimulate distinct APCs maturation and antigen presentation to T lymphocytes. Notably, high levels of antigen-specific CD8⁺ T cell responses, accompanied by the induction of CD4⁺ T helper cells, are achieved. More importantly, NVscp significantly increase the ratios of intratumoral CD8⁺ T/regulatory T cells and achieve prominent tumor therapy effects. The NOX2-inspired biomineralized NVscp represent an effective and easily applicable strategy that enables the strong cross-presentation of exogenous vaccine antigens.
... In addition, we used a multifunction microplate reader to identify the lipid oxidation level. In this study, BODIPY C11 was chosen because it could specifically monitor the ROS levels in membranes and lipids (Cheloni and Vi 2013). The BODIPY C11 results show us that erastin causes lipid peroxidation compared with control cells, whereas propofol and fer-1 significantly inhibited lipid peroxidation (Fig. 2C). ...
Propofol is a short-acting intravenous anesthetic that is widely used in clinical treatment. Previous articles have indicated that propofol is a therapeutic target for anti-apoptosis, anti-inflammation, anti-lipid peroxidation, and anti-reactive oxygen species (ROS). Moreover, cell ferroptosis is strongly correlated with cellular ROS, inflammatory responses, and lipid peroxidation. However, the mechanisms by which propofol attenuates neuronal injury by reducing ferroptosis remain unknown. Hence, we hypothesized that propofol could protect neurons by reducing ferroptosis. To test this hypothesis, HT-22 cells were treated with a specific ferroptosis activator (erastin) in the presence of propofol (50 μM). We found that propofol reduced erastin-induced high Fe²⁺ concentrations, lipid peroxides, and excess ROS. Western blotting results also suggested that propofol could rescue erastin-induced low expression of GXP4 and system Xc⁻. Further experiments indicated that propofol attenuated p-ALOX5 expression at Ser663 independent of ERK. In addition, we built two transient transfection cell lines, ALOX5 OE and Ser663Ala-ALOX5 OE, to confirm the target of propofol. We found that the Ser663 point is the critical role of propofol in rescuing erastin-induced cell injury/lipid peroxidation. In conclusion, propofol may help attenuate ferroptosis, which may provide a new therapeutic method to treat neuronal injury or the brain inflammatory response.
... T. thermophile regulates its membrane's lipid composition to more rigid configuration by lowering its unsaturated fatty acids and increasing saturated fatty acids. Studies also evidence the lipid peroxidation in rainbow and C. reinhardtii when exposed to CuO NPs [58,59]. The studies also report cytotoxicity of CuO NPs in primary liver cells of cat fish and HepG2 due to generation of ROS [60,61]. ...
Copper oxide nanoparticles (CuO NPs) use has exponentially increased in various applications (such as industrial catalyst, gas sensors, electronic materials, biomedicines, environmental remediation) due to their flexible properties, i.e. large surface area to volume ratio. These broad applications, however, have increased human exposure and thus the potential risk related to their short- and long-term toxicity. Their release in environment has drawn considerable attention which has become an eminent area of research and development. To understand the toxicological impact of CuO NPs, this review summarises the in-vitro and in-vivo toxicity of CuO NPs subjected to species (bacterial, algae, fish, rats, human cell lines) used for toxicological hazard assessment. The key factors that influence the toxicity of CuO NPs such as particle shape, size, surface functionalisation, time–dose interaction and animal and cell models are elaborated. The literature evidences that the CuO NPs exposure to the living systems results in reactive oxygen species generation, oxidative stress, inflammation, cytotoxicity, genotoxicity and immunotoxicity. However, the physio-chemical characteristics of CuO NPs, concentration, mode of exposure, animal model and assessment characteristics are the main perspectives that define toxicology of CuO NPs.
... Hg also increases the production of reactive oxygen species (ROS) that might result in oxidative stress, and is evidenced by the alteration in the activity of enzymes involved in ROS regulation, such as catalases, class III peroxidases (POD), lipoxygenases, or superoxide dismutases (SOD) [30,31]. SOD, POD and catalase activities were induced by 5 µM IHg 7d in Lemna minor [22] and excessive ROS generation was evidenced in Chlamydomonas reinhardtii exposed to 1 × 10 −7 M IHg or 1 × 10 −9 M MMHg [32] or for 96 h to 2 × 10 −6 M IHg [33], and resulting in lipid peroxidation after 24 h exposure to 5 × 10 −6 M IHg [34]. In E. nuttallii, after 24 h exposure to 4 × 10 −10 M and 4 × 10 −5 M IHg, SOD and POD activities showed a 1.3× and 1.3× increase and a 2.1× and 4.6× decrease, respectively [35] (Table 1). ...
(1) Background: Mercury is a threat for the aquatic environment. Nonetheless, the entrance of Hg into food webs is not fully understood. Macrophytes are both central for Hg entry in food webs and are seen as good candidates for biomonitoring and bioremediation; (2) Methods: We review the knowledge gained on the uptake and effects of inorganic Hg (IHg) and methyl-Hg (MMHg) in the macrophyte Elodea nuttallii found in temperate freshwaters; (3) Results: E. nuttallii bioaccumulates IHg and MMHg, but IHg shows a higher affinity to cell walls. At the individual level, IHg reduced chlorophyll, while MMHg increased anthocyanin. Transcriptomics and metabolomics in shoots revealed that MMHg regulated a higher number of genes than IHg. Proteomics and metabolomics in cytosol revealed that IHg had more effect than MMHg; (4) Conclusions: MMHg and IHg show different cellular toxicity pathways. MMHg’s main impact appears on the non-soluble compartment, while IHg’s main impact happens on the soluble compartment. This is congruent with the higher affinity of IHg with dissolved OM (DOM) or cell walls. E. nuttallii is promising for biomonitoring, as its uptake and molecular responses reflect exposure to IHg and MMHg. More generally, multi-omics approaches identify cellular toxicity pathways and the early impact of sublethal pollution.
... Lipid peroxidation occurs when polyunsaturated fatty acids are subject to oxidation by peroxyl radicals (Ayala et al., 2014); these can be quantified using the BODIPY reagent (Cheloni et al., 2013). BODIPY contains a polyunsaturated butadienyl region that is oxidized in the presence of ROS, resulting in a fluorescence shift. ...
New molecules that kill cancer cells by alternate mechanisms are urgently required to provide additional strategies for the treatment of tumours that are resistant to current drugs. Recent evidence from the Cancer Pharmacogenomics and Drug Development research group has indicated that an active polyunsaturated fatty acid metabolite, omega-3-epoxy-eicosapentaenoic acid (ω-3-epoxy-EPA), selectively inhibits tumour cell proliferation and activates apoptosis. Most recently a series of ω-3-epoxy-EPA (aryl urea fatty acid) derivatives were prepared and found to be more active than ω-3-epoxy-EPA itself in the killing of aggressive human triple-negative MDA-MB-231 breast cancer cells.
To date several of these agents have been found to effectively kill MDA-MB-231 cells in vitro and in vivo in xenografted nude mice. To optimize these molecules for eventual clinical application it is necessary to understand their anticancer mechanisms.
The aim of this project was to investigate the mechanisms of anticancer drug action of novel aryl urea fatty acid analogues. Techniques utilized in this study include cell culture, cell-based assays for the assessment of cancer cell viability (energy metabolism, reactive oxygen species production analysis via flow cytometry, and cell death assays), protein immunoblotting, and RNA-seq and real-time RT-PCR to evaluate altered gene expression.
Aryl urea fatty acid analogues were found to be active across a series of breast cancer cell lines reflecting different cancer subtypes. The information presented in this present thesis could be used to further develop aryl urea fatty acids as potential agents for the treatment of cancer.
... Lipid peroxidation was visualized using the fluorescent dye 4,4difluoro-5-(4-phenyl-1,3-butadienyl)-4-bora-3a,4a-diaza-s-indacene-3undecanoicacid (C11-BODIPY 581/591 , Invitrogen), which applicability in microalgae was demonstrated by Almeida et al. (2017) and Cheloni and Slaveykova (2013). Algal cells (1 × 10 6 mL − 1 ) were suspended in 100 mmol L − 1 PBS (pH 7.0) and incubated, in the dark, with C11-BOD-IPY 581/591 in a final concentration of 5 μmol L − 1 , for 4 h. ...
This study investigated the effect of the herbicide metolachlor (MET) on the redox homeostasis of the freshwater green alga Pseudokirchneriella subcapitata. At low MET concentrations (≤40 μg L-1), no effects on algal cells were detected. The exposure of P. subcapitata to 45-235 μg L-1 MET induced a significant increase of reactive oxygen species (ROS). The intracellular levels of ROS were particularly increased at high (115 and 235 μg L-1) but environmentally relevant MET concentrations. The exposure of algal cells to 115 and 235 μg L-1 MET originated a decrease in the levels of antioxidants molecules (reduced glutathione and carotenoids) as well as a reduction of the activity of scavenging enzymes (superoxide dismutase and catalase). These results suggest that antioxidant (non-enzymatic and enzymatic) defenses were affected by the excess of MET. As consequence of this imbalance (ROS overproduction and decline of the antioxidant system), ROS inflicted oxidative injury with lipid peroxidation and damage of cell membrane integrity. The results provide further insights about the toxic modes of action of MET on a non-target organism and emphasize the relevance of toxicological studies in the assessment of the impact of herbicides in freshwater environments.
... It has been shown in the literature that ROS can cause lipid peroxidation in cell membranes (Almeida et al. 2019). An increase in ROS resulted in a significant increase in lipid peroxidation levels and indicated an increase in the production of oxyl-radicals such as HO %, ROO %, RO % and ONOO- (Cheloni and Slaveykova 2013). In photosynthetic organisms, ROS can also damage lipid compounds of chloroplast thylakoid membranes such as MGDG, DGDG, SQDG and PG (Almeida et al. 2019). ...
Fatty acids (FA), which are the main components of storage and structural lipids, are crucial for the maintenance of membrane fluidity and play a central role in metabolic energy storage. Pesticide impacts on FA profiles have been documented in marine microalgae, but remain understudied in freshwater diatoms. In the same way, no study has focused on the impact of herbicides on their lipid content. The main aims of this study were to: 1) compare the responses of FA profiles and “classical descriptors” in order to evaluate their complementarity, 2) estimate the effect of herbicides on lipid content and composition of one freshwater diatom and finally, 3) examine the potential of 20:5n3 as a biomarker of herbicide pollution. To address these issues, Gomphonema gracile and Nitzschia palea were exposed during seven days to diuron and S-metolachlor at 10 μg.L-1. The results demonstrated higher toxicity of diuron compared to S-metolachlor at 10 μg.L-1 on all descriptors studied. FA responded to diuron contamination conversely to pigment content and permitted to document energy content variations in diatoms subjected to herbicide stress conversely to “classical descriptors”. Total lipid composition was impacted by diuron with a notable decrease of storage lipids. Diuron affected the content of 20:5n3 in lipids involved in photosynthesis. Based on previous studies and our current findings, it is suggested that 20:5n3 content in diatoms could be a good biomarker of diuron pollution in freshwaters.
... The intensity of orange, green, and red autofluorescence can vary in response to changes in the physiological state of microalgae cells under various oxidative stress conditions (Ying and Dobbs, 2007). According to the above findings, Cheloni and Slaveykova (2013) reported also a change in the autofluorescence of C. reinhardtii exposed to copper (Cu(II)), Hg(II), and nanoparticulate Cu(II) oxide. ...
This study highlights the mechanisms of Pb(II)-phycoremediation using the Pb(II) tolerant strain of Scenedesmus obliquus. First, monitoring of cell growth kinetics in control and Pb(II)-doped medium revealed significant growth inhibition, while the analyses through flow cytometry and Zetasizer revealed no difference in cell viability and size. Residual weights of control and Pb(II)-loaded cells assessed by thermogravimetric analysis were 31.34% and 57.8%, respectively, indicating the uptake of Pb(II) into S. obliquus cells. Next, the use of chemical extraction to distinguish between the intracellular and extracellular uptake indicated the involvement of both biosorption (85.5%) and bioaccumulation (14.5%) mechanisms. Biosorption interaction of Pb(II) ions and the cell wall was confirmed using SEM-EDX, FTIR, zeta potential, zero-charge pH, and contact angle analyses. Besides, the biochemical characterization of control and Pb(II)-loaded cells revealed that the bioaccumulation of Pb(II) induces significant increases in the carotenoids and lipids content, while it decreases in the chlorophyll, carbohydrates, and proteins content. Finally, the metabolomic analysis indicated an increase in the relative abundance of fatty acid methyl esters, alkanes, aromatic compounds, and sterols. However, the alkenes and monounsaturated fatty acids decreased. Such metabolic adjustment may represent an adaptive strategy that prevents high Pb(II)-bioaccumulation in cellular compartments.
... The characteristics of eryptosis and corresponding investigation methods[144,[203][204][205][206][207][208][209][210][211] ...
Background:
Renal anemia is an important complication of chronic kidney disease (CKD). In addition to insufficient secretion of erythropoietin (EPO) and erythropoiesis disorders, the impact of eryptosis on renal anemia demands attention. However, a systemic analysis concerning the pathophysiology of eryptosis has not been expounded.
Summary:
The complicated conditions in CKD patients, including oxidative stress, osmotic stress, metabolic stress, accumulation of uremic toxins, and iron deficiency, affects the normal skeleton structure of red blood cells (RBCs) and disturbs ionic homeostasis, causing phosphatidylserine to translocate to the outer lobules of the RBC membrane that leads to early elimination and/or shortening of the red blood cell lifespan (RBCL). Inadequate synthesis of RBCs cannot compensate for their accelerated destruction, thus exacerbating renal anemia. Meanwhile, EPO treatment alone will not reverse renal anemia. A variety of eryptosis inhibitors have so far been found, but evidence of their effectiveness in the treatment of CKD remains to be established. Key Messages: In this review, the pathophysiological processes and factors influencing eryptosis in CKD were elucidated. The aim of this review is to underline the importance of eryptosis in renal anemia and determine some promising research directions or possible therapeutic targets to correct anemia in CKD.
... BODIPY C-11 581/591 (undecanoic acid) was used for the analysis of lipid peroxidation. This probe detects the oxidation of the polyunsaturated butadienyl portion, changing its emission peak from ∼590 nm to ∼510 nm (Cheloni and Slaveykova, 2013). A 2-mM (1000X) BODIPY stock was prepared and frozen at −20°C. ...
Ferroptosis is an oxidative and iron-dependent form of regulated cell death (RCD) recently described in eukaryotic organisms like animals, plants, and parasites. Here, we report that a similar process takes place in the photosynthetic prokaryote Synechocystis sp. PCC 6803 in response to heat stress. After a heat shock, Synechocystis sp. PCC 6803 cells undergo a cell death pathway that can be suppressed by the canonical ferroptosis inhibitors, CPX, vitamin E, Fer-1, liproxstatin-1, glutathione (GSH), or ascorbic acid (AsA). Moreover, as described for eukaryotic ferroptosis, this pathway is characterized by an early depletion of the antioxidants GSH and AsA, and by lipid peroxidation. These results indicate that all of the hallmarks described for eukaryotic ferroptosis are conserved in photosynthetic prokaryotes and suggest that ferroptosis might be an ancient cell death program.
The present article reviews current knowledge and recent progress on the bioavailability and toxicity of mercury to aquatic primary producers. Mercury is a ubiquitous toxic trace element of global concern. At the base of the food web, primary producers are central for mercury incorporation
into the food web. Here, the emphasis is on key, but still poorly understood, processes governing the interactions between mercury species and phytoplankton, and macrophytes, two representatives of primary producers. Mass transfer to biota surface, adsorption to cell wall, internalization
and release from cells, as well as underlying toxicity mechanisms of both inorganic mercury and methylmercury are discussed critically. In addition, the intracellular distribution and transformation processes, their importance for mercury toxicity, species-sensitivity differences and trophic
transfer are presented. The mini-review is illustrated with examples of our own research.
Cell cycle studies in plants and algae are highly dependent on reliable methods for detecting cellular DNA replication. With its short growth cycle and ease of genetic transformation, Phaeodactylum tricornutum is an important model organism for the study of pennate diatoms. Here we explored two different methods to detect the cell cycle of P. tricornutum, one using SYBR‐green I to via flow cytometry, and the other using EdU labeling to observe cell cycle changes under fluorescence microscopy. Both EdU labeling fluorescence microscopy and SYBR‐green I staining flow cytometry accurately indicated that the cells of P. tricornutum enter the G2/M phase after 12 h of light exposure. The results indicate that SYBR Green I was an adequate detection method for nuclear DNA quantitation in cells of P. tricornutum using a flow cytometer and without RNase A treatment. In addition, EdU can be applied to P. tricornutum to reliably detect cell proliferation. Besides, Mg‐ProtoIX was able to reverse the cell cycle division inhibition of P. tricornutum and allow the nuclear DNA replication to proceed normally. Taken together, the photoperiodic division time point was clearly identified, which sheds light on the regulation of cell division mechanism in P. tricornutum.
The effects of short-term exposure to sub-nanomolar methyl-mercury (MeHg) concentrations, representative of contaminated environments, on the microalga Chlamydomonas reinhardtii were assessed using both physiological endpoints and gene expression analysis. MeHg bioaccumulated and induced significant increase of the photosynthesis efficiency, while the algal growth, oxidative stress and chlorophyll fluorescence were unaffected. At the molecular level, MeHg significantly dysregulated the expression of genes involved in motility, energy metabolism, lipid metabolism, metal transport and antioxidant enzymes. Data suggest that the cells were able to cope with sub-nanomolar MeHg exposure, but this tolerance resulted in a significant cost to the cell energy and reserve metabolism as well as ample changes in the nutrition and motility of C. reinhardtii. The present results allowed gaining new insights on the effects and uptake mechanisms of MeHg at sub-nanomolar concentrations in aquatic primary producers.
Graphene quantum dots (GQDs) are an excellent tool for theranostics, and are widely used in nanomedical applications. The biosafety of GQDs has received abundant attention, but their latent toxicological mechanisms remain inadequately understood. To investigate the cellular and molecular mechanisms underlying graphene-mediated changes, quantitative proteomics and untargeted lipidomics were integrated. We discovered that glutathione peroxidase 4 as a key regulator of ferroptosis, was down-regulated at the protein level by GQDs. Lipidomics profiling with features of ferroptosis was identified in GQDs-treated RAW264.7 macrophages. Furthermore, GQDs exposure was associated with reduced levels of GSH and increased lipid peroxidation. Overexpression of GPX4 in RAW264.7 cells and pre-treatment of a ferroptosis inhibitor Ferrostatin-1 (Fer-1) not only suppressed cell death, but also alleviated lipid peroxidation. Taken together, our results indicated that GQDs exposure induced ferroptosis in RAW264.7 macrophages, and provided essential data for biosafety evaluations of GQDs.
1-Aminocyclopropane-1-carboxylate (ACC) deaminase is a well-known bacterial producing enzyme that helps plants to overcome stress conditions by modulating ethylene biosynthesis. However, the functional role of ACC deaminase and ethylene in microalgae during stress remains to be explored. In this study, to investigate the role of ACC deaminase (acds) from Pseudomonas putida UW4 in enhancing the biomass and lipid content of Chlamydomonas under nitrogen deficit condition. The synthetic codon-optimized acds gene was cloned into vector pChlamy_4 and introduced into Chlamydomonas. Results indicated that Chlamydomonas-expressing acds lines showed significant tolerance to nitrogen-deficit by reducing the ethylene content. The biomass, chlorophyll content and photosynthetic activity of acds-expressing lines were significantly increased during nitrogen deficit condition. Moreover, the intracellular lipid and fatty acid content were much higher in acds-expressing lines than the wild-type. In terms of stress alleviation, the transgenic lines displayed increased antioxidant enzymes, reduced ROS and lipid peroxidation levels.
Oxidative stress has been implicated in cellular senescence and aging, as well as in the onset and progression of many diverse genetic and acquired diseases and conditions. However, reactive oxygen (ROS) and nitrogen (RNS) species initiating oxidative stress also serve important regulatory roles, mediated by intercellular and intracellular signaling, adaptation to endogenous and exogenous stress, and destruction of invading pathogens. Fluorescence-based analysis of oxidative stress and related processes is an important cytomic application; almost 4000 papers were published between 1989 and 2016. To ascertain the specific role of ROS and RNS in oxidative stress studies by cytomic methodologies, it is essential to detect and characterize these species accurately. Unfortunately, the detection and quantitation of individual intracellular ROS and RNS remains a challenge, but different, complementary cytometric strategies directed toward other endpoints of oxidative stress may also be considered. In this chapter we present and briefly discuss the limitations and perspectives of such approaches.
The lipid membrane is a fundamental part of life. However, the effects of different stresses on membranal integrity and physiology are less understood. Using novel 4-aminophthalimide-based membrane-specific dyes (4AP-Cn: n is carbon chain-length), aided with confocal microscopy, fluorescence spectroscopy, molecular dynamics simulations, and flow cytometry, we have studied stress-mediated changes in E. coli membranes. By exploiting the depth-dependent positioning and subsequent environmental sensitivity of the dyes, we have proposed a measure of antibiotic-induced membrane damage: the fluorescence Peak Maxima Difference (PMD) between 4AP-C9 and 4AP-C13. The ROS-influenced PMD quantifies cytoplasmic membrane thickness and measures sensitivity against most bactericidal antibiotics, depending upon the extent of lipid peroxidation. Importantly, we have verified this observation using antibiotic-sensitive and resistant clinical isolates of E. coli and ESKAPE pathogens like K. pneumoniae and Enterobacter spp. This study underscores the role of antibiotic-induced membrane changes; a critical issue given the increasing global antimicrobial resistance.
In photosynthetic microorganisms, the toxicity of carbon nanomaterials (CNMs) is typically characterized by a decrease in growth, viability, photosynthesis, as well as the induction of oxidative stress. However, it is currently unclear how the shape of the carbon structure in CNMs, such as in the 1-dimensional carbon nanotubes (CNTs) compared to the two-dimensional graphene oxide (GO), affects the way they interact with cells. In this study, the effects of GO and oxidized multi-walled CNTs were compared in the cyanobacterium Microcystis aeruginosa to determine the similarities or differences in how the two CNMs interact with and induce toxicity to cyanobacteria. Using change in Chlorophyll a concentrations, the effective concentrations inducing 50% inhibition (EC50) at 96 h are found to be 11.1 μg/mL and 7.38 μg/mL for GO and CNTs, respectively. The EC50 of the two CNMs were not found to be statistically different. Changes in fluorescein diacetate and 2′,7′-dichlorodihydrofluorescein diacetate fluorescence, measured at the EC50 concentrations, suggest a decrease in esterase enzyme activity but no oxidative stress. Scanning and transmission electron microscopy imaging did not show extensive membrane damage in cells exposed to GO or CNTs. Altogether, the decrease in metabolic activity and photosynthetic activity without oxidative stress or membrane damage support the hypothesis that both GO and CNTs induced indirect toxicity through physical mechanisms associated with light shading and cell aggregation. This indirect toxicity explains why the intrinsic differences in shape, size, and surface properties between CNTs and GO did not result in differences in how they induce toxicity to cyanobacteria.
Oxygen evolution complex (OEC) in photosystem II (PSII) is sensitive to environmental stressors. However, oxidative damage mechanism in PSII-OEC is still unclear. Here, we investigated photosynthetic performance of PSII, oxidative stress and antioxidant reaction induced by reactive oxygen species (ROS) in a unicellular green alga Chlorella vulgaris (C. vulgaris) under the stress of cetyltrimethylammonium chloride (CTAC). From the changes of chlorophyll fluorescence parameters and PSII activity, it was proved that the electron transport, which occurred initially at the electron donor side of OEC, was disturbed by CTAC. Moreover, a significant decrease of the oxygen evolution rate in OEC (40.95%) while an increase of ROS (50.50%) was obtained after the exposure to 0.6 mg/L CTAC compared to the control (without CTAC), confirming that more oxygen transferred to ROS under the stress. Furthermore, the increased ROS in chloroplast and the structural destruction in thylakoid membrane were observed, respectively. These results proved that oxidative damage mechanism in PSII-OEC is mainly through the reduction of oxygen evolution and the production of excessive ROS, thus leading to the destruction of OEC performance and chloroplast structure.
Renal ischemia/reperfusion injury (IRI) is a significant problem in perioperative
medicine related to oxidative programmed cell death. However, the role of ferroptosis,
a newly discovered form of oxidative cell death, has not been evaluated widely.
Panx1, an ATP-releasing pathway family, has anti-inflammatory effects during kidney
injury. Here, we demonstrated that panx1 deletion protected against renal IRI by
regulating ferroptotic cell death. In panx1 knockout mice subjected to renal IRI,
decreased plasma creatinine, MDA contents in kidney tissue, and tubular cell death
(decreased TUNEL positive renal tubular cells) were found compared to those in
panx1 wild type (wt) mice. In cultured human kidney 2 (HK-2) cells, erastin induced
ferroptotic lipid peroxidation and iron accumulation were significantly attenuated by
silencing panx1 expression. Moreover, panx1 silencing significantly modulated
ferroptosis related protein expression. Furthermore, Panx1 deletion induced the
expression of a cytoprotective chaperone, heme oxygenase-1 (HO-1), and
modulated the MAPK/ERK pathway. In this study, we first discovered that panx1
deletion protects against renal IRI by attenuating MAPK/ERK activation and inducing
HO-1 expression in a ferroptotic way. Our findings provide new insights to
understand the mechanism of panx1 in ferroptotic cell death and highlight a new
therapeutic target for acute kidney injury(AKI) treatment during the perioperative
period.
Nanotechnology is a major newfound scientific growth area that can exhibit a variety of risks for environments. The small size of nanoparticles and their morphology, as well as their surface properties, provide surfaces that can bind and carry toxic chemical contaminants. For hazard evaluation of nanoparticles, quantitative ecotoxicological data are required. This chapter presents a complete literature review on the toxicity (L(E) C50 values) of nanoparticles (NPs) to ecological receptors such as algae and aquatic plants (1) to recognize the most harmful nanoparticles and sensitive organism classes; (2) to identify the biological characteristics of the (photocatalytic) nanoparticles that have the most biological importance; and (3) to provide accordant ecotoxicological information for further hazardous assessment. The focus was assessed on some synthetic nanoparticles and organism groups representing main food hain levels (algae and aquatic plants). Furthermore, this chapter provides an extensive literature overview on Ag, CuO and ZnO, and TiO2 NPs toxicity mechanisms on the basis of different environmentally accordant test species in vitro and factors to modify the ecotoxic effect of NPs. Photo-induced toxicity can be another considerable mechanism of toxicity under environmentally accordant UV irradiation. However, the usual rarity of experimental data, their disparate distribution among the photocatalytic nanoparticles and environmental states, the difficulties in modifying nanoparticles and obtaining homogeneous and stable suspensions, and the confusion from indistinct metrics, are discussed.
Drying processes do not eliminate pathogenic Escherichia coli in foods but induce sublethal injury, which may also induce the Shiga toxin (Stx) prophage. This study investigated the effect of drying on membrane lipid oxidation and stx expression in E. coli. Lipid peroxidation was probed with C11-BODIPY581/591; and stx expression was assayed by quantification of GFP in E. coli O104:H4 Δstx2a:gfp:ampr. Treatment of E. coli with H2O2 oxidized the probe; probe oxidation was also observed after drying and rehydration. Lipid oxidation and the lethality of drying were reduced when cells were dried with trehalose under anaerobic condition; in addition, viability and probe oxidation differed between E. coli AW1.7 and E. coli AW1.7Δcfa. Desiccation tolerance thus relates to membrane lipid oxidation. Drying also resulted in expression of GFP in 5% of the population. Overexpression of gfp and recA after drying and rehydration suggested that the expression of Stx prophage was regulated by the SOS response. Overall, C11-BODIPY581/591 allowed investigation of lipid peroxidation in bacteria. Drying causes lipid oxidation, DNA damage and induction of genes encoded by the Stx prophage in E. coli.
Heavy metals, depending on their oxidation states, can be highly reactive and, as a consequence, toxic to most organisms. They are produced by an expanding variety of anthropogenic sources suggesting an increasingly important role for this form of pollution. The toxic effect of heavy metals appears to be related to production of reactive oxygen species (ROS) and the resulting unbalanced cellular redox status. Algae respond to heavy metals by induction of several antioxidants, including diverse enzymes such as superoxide dismutase, catalase, glutathione peroxidase and ascorbate peroxidase, and the synthesis of low molecular weight compounds such as carotenoids and glutathione. At high, or acute, levels of metal pollutants, damage to algal cells occurs because ROS levels exceed the capacity of the cell to cope. At lower, or chronic, levels algae accumulate heavy metals and can pass them on to organisms of other trophic levels such as mollusks, crustaceans, and fishes. We review here the evidence linking metal accumulation, cellular toxicity, and the generation of ROS in aquatic environments.
: The generation of reactive oxygen species (ROS) and scavenging of the superoxide radical by superoxide dis-mutase (SOD) was studied in mat-like canopies of the green macroalga Ulva rotundata Bliding in a tidal brine pond system in southern Spain. Artificial canopies were covered with different cut-off filters, generating different radiation conditions. ROS and SOD were assessed after three days of exposure. ROS induced lipid peroxidation depended on the position of indi-vidual thalli within the canopy and on radiation conditions. Samples exposed to the full solar spectrum were most affected, whereas samples either exposed to photosynthetically active radiation (PAR) alone or UV radiation without PAR exhibited fewer peroxidation products. The activity of SOD appeared to be controlled by the impinging UV-A and UV-B radiation and also increased in response to oxidative stress. The results provide evidence for additive effects of high PAR and UV-B under field conditions and support the previously proposed hypothesis that UV-B effects are mediated by an inhibition of the xanthophyll cycle, which increases ROS production and, consequently, causes oxidative damage to components of the photosynthetic machinery, such as proteins and pigments.
We used the flow sorting capacities of a benchtop FACSCalibur flow cytometer to analyze the phytoplankton community of four different aquatic ecosystems. We show that despite the high optical, mechanistic, and hydrodynamic stress for the cells while sorted, most of the targeted populations could be isolated and grew in mixed culture media subsequent to sorting. Forty-five phytoplankton taxa were isolated, including green algae (29 species), cyanobacteria (eight), diatoms (seven), and cryptomonads (one). The isolation success average was high since 80% of the total sorted populations grew successfully and 47% constituted monocultures. It is noteworthy, however, that some groups could not be isolated, as for example colonial cyanobacteria, chrysophytes, euglenophytes, desmids, or dinoflagellates, and some species such as Cryptomonas sp. were very sensitive to the sorting process. It is proposed that flow cytometric analysis of freshwater phytoplankton
might be a relevant tool for water managers and could be applied in some specific cases, such as early monitoring of blooming taxa or basic bio-monitorings of key species. The higher isolation average obtained from the flow sorting can also be powerful for the physiological or molecular study of some taxa after their cultivation.
The current interest in microalgae as a sustainable source of next generation biofuels and other valuable substances is driving exploration of their use as unique biotechnological production systems. To design and optimise appropriate production strategies, the behaviour of particular microalgal species should be well characterised under different culture conditions. Thus, flow cytometric (FCM) methods, which are already well established in environmental and toxicological studies of microalgae, are also useful for analysing the physiological state of microalgae, and have the potential to contribute to the rapid development of feasible bioprocesses. These methods are commonly based on the examination of intrinsic features of individual cells within a population (such as autofluorescence or size). Cells possessing the desired physiological or morphological features, which are detectable with or without fluorescent staining, are counted or isolated (sorted) using an FCM device. The options for implementation of FCM in the development of biotechnological processes detailed in this review are (i) analysing the chemical composition of biomass, (ii) monitoring cellular enzyme activity and cell viability, and (iii) sorting cells to isolate those overproducing the target compound or for the preparation of axenic cultures.
Freshwater microalga Chlorella vulgaris and marine microalga Dunaliella tertiolecta were used to investigate toxic effects induced by 50 nm silver nanoparticles (AgNPs). To induce AgNPs effect, we exposed Chlorella vulgaris and Dunaliella tertiolecta for 24h to 0-10 mg/L. We showed that growth media had different effects in AgNPs agglomerates' formation. Cellular viability, reactive oxygen species (ROS) formation and lipids peroxidation were employed to assess the toxic effects of AgNPs. AgNPs were able to interact directly with the Chlorella vulgaris cells surface and large aggregates were observed. AgNPs have a negative effect on Chlorella vulgaris and Dunaliella tertiolecta, as manifested by a strong decrease in chlorophyll content, viable algal cells, increased ROS formation and lipids peroxidation. The variability in sensitivity of both algae towards AgNPs was observed. We conclude that AgNPs have a negative effect on aquatic algae and these alterations might have serious consequences on structure and function of aquatic plant communities.
Investigation of mercury toxicology in green algae is of great importance from ecological point of view, because mercury has become a major contaminant in recent years. In higher plants, accumulation of mercury modifies many aspects of cellular functions. However, the process that mercury exerts detrimental effects on green algae is largely unknown. In this study, we performed an experiment focusing on the biological responses of Chlamydomonas reinhardtii, a unicellular model organism, to Hg(2+)-induced toxicity. C. reinhardtii was exposed to 0, 1, 2, 4, 6, and 8 μM Hg in media. Concentrations of Hg were negatively correlated with the cell growth. Treatment with Hg induced accumulation of reactive oxygen species and peroxidative products. Endogenous proline levels increased in Hg-exposed algae. Hg exposure activated superoxide dismutase (SOD), catalase (CAT) and ascorbate peroxidase (APX). To get insights into the molecular response, a RT-PCR-based assay was performed to analyze the transcript abundance of Mn-SOD, CAT and APX. Our analysis revealed that expression of the genes was up-regulated by Hg exposure, with a pattern similar to the enzyme activities. Additional investigation was undertaken on the effect of Hg on the transcript amount of ∆(1)-pyrroline-5-carboxylate synthetase, a key enzyme of proline biosynthesis and on that of heme oxygenase-1 (HO-1), an enzyme regulating heavy metal tolerance. Expressions of both P5CS and HO-1 were up-regulated by Hg. These data indicate that Hg-induced oxidative stress was responsible for the disturbance of the growth and antioxidant defensive systems in C. reinhardtii.
Lipid peroxidation (LPO) of stallion spermatozoa was assessed in fresh semen and in samples of the same ejaculates after freezing and thawing. Particular attention was paid to individual differences in the susceptibility to LPO and its possible relationship with freezability. Innate levels of LPO were very low in fresh spermatozoa but increased after thawing, a change that was largely stallion-dependent. The level of LPO in fresh spermatozoa was not correlated with that of the thawed spermatozoa. Negative correlations existed between LPO and intact membranes post-thaw (r=-0.789, P<0.001), and also between LPO and spermatozoa with high mitochondrial membrane potential (Deltapsim) post-thaw (r=-0.689, P<0.001). LPO was also highly and significantly correlated with caspase activity. The correlation between caspase activity in ethidium positive cells and LPO was r=0.772, P<0.001. This LPO is unlikely to represent, per se, a sign of cryopreservation-induced injury, but it is apparently capable of triggering 'apoptotic-like changes' that could result in the sub-lethal cryodamage often seen among surviving spermatozoa.
Reactive oxygen species (ROS) and other radicals are involved in a variety of biological phenomena, such as mutation, carcinogenesis, degenerative and other diseases, inflammation, aging, and development. ROS are well recognized for playing a dual role as deleterious and beneficial species. The objectives of this review are to describe oxidative stress phenomena, terminology, definitions, and basic chemical characteristics of the species involved; examine the biological targets susceptible to oxidation and the defense mechanisms of the organism against these reactive metabolites; and analyze methodologies, including immunohistochemical markers, used in toxicological pathology in the visualization of oxidative stress phenomena. Direct detection of ROS and other free radicals is difficult, because these molecules are short-lived and highly reactive in a nonspecific manner. Ongoing oxidative damage is, thus, generally analyzed by measurement of secondary products including derivatives of amino acids, nuclei acids, and lipid peroxidation. Attention has been focused on electrochemical methods based on voltammetry measurements for evaluating the total reducing power of biological fluids and tissues. This approach can function as a tool to assess the antioxidant-reducing profile of a biological site and follow changes in pathological situations. This review thus includes different topics essential for understanding oxidative stress phenomena and provides tools for those intending to conduct study and research in this field.
Short-term uptake of cadmium by a wild-type (2137) and a cell wall-less strain (CW-2) of Chlamydomonas reinhardtii was examined as a function of Cd speciation in a well-defined, aqueous medium. Internalization fluxes were determined for free cadmium concentrations ranging from 5 x 10(-10) M to 5 x 10(-4) M in the presence of ligands forming both labile and inert hydrophilic complexes. A first-order biological internalization, as predicted by the free ion activity model (FIAM), was observed for both strains. The maximum Cd internalization flux, Jmax, for the wild-type strain was 5-fold higher (1.3 x 10(-11) mol cm(-2) min(-1)) than for the CW-2 strain (2.3 x 10(-12) mol cm(-2) min(-1)) and was not influenced by the presence of competitors such as Ca in the experimental solution. The conditional stability constant for the adsorption of Cd to transport sites of the CW-2 strain was 5-fold higher (10(6.7) M(-1)) than for the wild-type strain (10(6) M(-1)). Competition experiments demonstrated that Mo, Mn, Cu, Co, Zn, Ni, Ca, and Pb inhibited, at least partially, Cd uptake, while no inhibition was observed for similar concentrations of Mg and Fe. The stability constant for the competitive binding of Ca to the Cd transport site was determined to be 10(4.5) M(-1). Cu and Zn competed with Cd uptake sites with stability constants of 10(5.6) and 10(5.2) M(-1), respectively. Protons also appeared to compete with Cd uptake sites as uptake could generally be predicted quantitatively in their presence. Finally, in the presence of low concentrations (<20 mg L(-1)) of Suwannee River fulvic acid, Cd internalization fluxes could be predicted from [Cd2+], in accordance with the FIAM.
Algae are exposed to elevated levels of heavy metals in water bodies generally for a long-term, and occasionally for a short-term duration. The present study deals with oxidative stress in Scenedesmus sp., commonly found in nutrient-rich freshwaters, during short- (6h) and long-term (7d) exposure to Cu(2+) and Zn(2+). The cells accumulated almost 2- and 4-times more Cu(2+) and Zn(2+) inside the cells during long-term than during short-term exposure to these metals. But the data on photosynthetic O(2) evolution and cell viability suggest that Scenedesmus sp. experienced lesser metal stress in long-term than in short-term experiment. Although malondialdehyde content was slightly higher in the long-term experiment, the amount produced by one unit intracellular metal was significantly lower than that in the short-term experiment. Superoxide dismutase activity of Scenedesmus sp. was >30% higher during long-term than during short-term exposure to Cu(2+) and Zn(2+). But, catalase and ascorbate peroxidase activities increased only at 2.5 microM Cu(2+) and 25 microM Zn(2+) when oxidative stress was mild, but were inhibited at 10 microM Cu(2+) under intense oxidative stress. Cu(2+) and Zn(2+) reduced glutathione reductase activity and total SH content of Scenedesmus sp. in both the experiments, with greater reduction occurring in the long-term experiment. The depletion of total thiol was positively related with the intracellular level of metals. Thiols might have helped Scenedesmus sp. in overcoming metal-induced oxidative stress, but depletion of thiol pool is known to make cells vulnerable to oxidative stress. The study suggests that antioxidant enzymes play a role only under mild oxidative stress. An increased accumulation of proline seems to be an important strategy for alleviating metal-induced oxidative stress in Scenedesmus sp. The study shows that Scenedesmus sp. could acclimatize during long-term exposure to toxic concentrations of the test metals.
The potential of oxygen free radicals and other reactive oxygen species (ROS) to damage tissues and cellular components, called oxidative stress, in biological systems has become a topic of significant interest for environmental toxicology studies. The balance between prooxidant endogenous and exogenous factors (i.e., environmental pollutants) and antioxidant defenses (enzymatic and nonenzymatic) in biological systems can be used to assess toxic effects under stressful environmental conditions, especially oxidative damage induced by different classes of chemical pollutants. The role of these antioxidant systems and their sensitivity can be of great importance in environmental toxicology studies. In the past decade, numerous studies on the effects of oxidative stress caused by some environmental pollutants in terrestrial and aquatic species were published. Increased numbers of agricultural and industrial chemicals are entering the aquatic environment and being taken up into tissues of aquatic organisms. Transition metals, polycyclic aromatic hydrocarbons, organochlorine and organophosphate pesticides, polychlorinated biphenyls, dioxins, and other xenobiotics play important roles in the mechanistic aspects of oxidative damage. Such a diverse array of pollutants stimulate a variety of toxicity mechanisms, such as oxidative damage to membrane lipids, DNA, and proteins and changes to antioxidant enzymes. Although there are considerable gaps in our knowledge of cellular damage, response mechanisms, repair processes, and disease etiology in biological systems, free radical reactions and the production of toxic ROS are known to be responsible for a variety of oxidative damages leading to adverse health effects and diseases. In the past decade, mammalian species were used as models for the study of molecular biomarkers of oxidative stress caused by environmental pollutants to elucidate the mechanisms underlying cellular oxidative damage and to study the adverse effects of some environmental pollutants with oxidative potential in chronic exposure and/or sublethal concentrations. This review summarizes current knowledge and advances in the understanding of such oxidative processes in biological systems. This knowledge is extended to specific applications in aquatic organisms because of their sensitivity to oxidative pollutants, their filtration capacity, and their potential for environmental toxicology studies.
Green autofluorescence (GAF) has been described in the short flagellum of golden and brown algae, the stigma of Euglenophyceae, and cytoplasm of different life stages of dinoflagellates and is considered by some researchers a valuable taxonomic feature for dinoflagellates. In addition, green fluorescence staining has been widely proposed or adopted to measure cell viability (or physiological state) in areas such as apoptosis of phytoplankton, pollutant stresses on algae, metabolic activity of algae, and testing treatment technologies for ships' ballast water. This paper reports our epifluorescence microscopic observations and quantitative spectrometric measurements of GAF in a broad phylogenetic range of microalgae. Our results demonstrate GAF is a common feature of dinoflagellates, diatoms, green algae, cyanobacteria, and raphidophytes, occurs in the cytoplasm and particularly in eyespots, accumulation bodies, spines, and aerotopes, and is caused by molecules other than chlorophyll. GAF intensity increased with time after cell death or fixation and with excitation by blue or UV light and was affected by pH. GAF of microalgae may be only of limited value in taxonomy. It can be strong enough to interfere with the results of green fluorescence staining, particularly when stained samples are observed microscopically. GAF is useful, however, for microscopic study of algal morphology, especially to visualize cellular components such as eyespots, nucleus, aerotopes, spines, and chloroplasts. Furthermore, GAF can be used to visualize and enumerate dinoflagellate cysts in marine and estuarine sediments in the context of anticipating and monitoring harmful algal blooms and in tracking potentially harmful dinoflagellates transported in ships' ballast tanks.
Lipid peroxidation is known to be a major factor in the aetiology of defective sperm function. Although biochemical assays for this process exist, they are relatively insensitive and require large numbers of spermatozoa; a condition that cannot be met with many infertility specimens. Recently, a new approach for monitoring peroxidative damage has been introduced, involving the probe BODIPY (581/591) C(11), which readily incorporates into cells and undergoes a spectral emission shift when attacked by reactive oxygen metabolites. We have examined the applicability of this probe as an indicator of oxidative stress in human sperm populations using flow cytometry as an end point. The measurement of peroxidation with BODIPY C(11) demonstrated significant dependence on the presence of a ferrous ion promoter (P < 0.001), which was significantly enhanced in sperm recovered from low-density Percoll fractions (P < 0.05) and was particularly damaging to the sperm midpiece. Iron-induced radical formation was suppressed by ascorbate in a dose-dependent manner (P < 0.001) and could only be promoted by Fe(II) and Cu(II); nickel, zinc and Fe(III) were ineffective. The Fe(II)-promoted BODIPY C(11) signal was significantly correlated with the measurement of reactive oxygen species generation with dihydroethidium. We conclude that BODIPY C(11) is an extremely useful probe for indexing peroxidative damage in human spermatozoa.
An imbalance between production of reactive oxygen species (ROS) and antioxidant defense is involved in the pathogenesis of diverse chronic parenchymatous diseases. To identify the primary site of such increased oxidative stress, a lipophilic ROS-sensitive probe (C11-Bodipy 581/591) is introduced, which allows the visualization and quantification of oxidative injury using confocal fluorescence microscopy in living cells. The properties of this probe are such that its emission wavelength irreversibly shifts from red to green upon oxidation. This probe was used to identify the spatiotemporal distribution of lipid peroxidation in the rat kidney during chronic NOS inhibition, a model associated with hypertension and proteinuria. Chronic NOS inhibition resulted in increased lipid peroxidation in renal tubules but hardly any in glomeruli or blood vessels. This peroxidation preceded the loss of renal function characteristic of the model and was accompanied by parallel changes in thiobarbituric acid reactive substances in the renal cortex. Furthermore, the increase in oxidation was dependent on angiotensin II and NADPH oxidase and prevented by vitamin E. Induction of cytoprotective heat-shock protein 70 preceded lipid peroxidation, rise in BP, or proteinuria. These findings challenge the paradigm that the vascular wall is the source and target of oxidative stress in chronic parenchymatous renal disease associated with hypertension.
High-resolution photomicrographs of phytoplankton cells and chains can now be acquired with imagingin-flow systems at rates that make manual identification impractical for many applications. To address the challenge for automated taxonomie identification of images generated by our custom-built submersible Imaging FlowCytobot, we developed an approach that relies on extraction of image features, which are then presented to a machine learning algorithm for classification. Our approach uses a combination of image feature types including size, shape, symmetry, and texture characteristics, plus orientation invariant moments, diffraction pattern sampling, and co-occurrence matrix statistics. Some of these features required preprocessing with image analysis techniques including edge detection after phase congruency calculations, morphological operations, boundary representation and simplification, and rotation. For the machine learning strategy, we developed an approach that combines a feature selection algorithm and use of a support vector machine specified with a rigorous parameter selection and training approach. After training, a 22-category classifier provides 88% overall accuracy for an independent test set, with individual category accuracies ranging from 68% to 99%. We demonstrate application of this classifier to a nearly uninterrupted 2-month time series of images acquired in Woods Hole Harbor, including use of statistical error correction to derive quantitative concentration estimates, which are shown to be unbiased with respect to manual estimates for random subsamples. Our approach, which provides taxonomically resolved estimates of phytoplankton abundance with fine temporal resolution (hours for many species), permits access to scales of variability from tidal to seasonal and longer. © 2007, by the American Society of Limnology and Oceanograpny, Inc.
Oxyfluorfen and diuron, herbicides commonly used in champagne vineyards, are known to generate reactive oxygen species (ROS) which induce a shift of the balance between prooxidative and antioxidative reactions. Against this oxidative stress, plants have developed enzymatic defenses such as superoxide dismutase, peroxidases, and catalase to limit lipid peroxidation, DNA damage, and protein degradation. These antioxidative enzyme activities were used as biomarkers in the present study to evaluate the toxic effect of oxyfluorfen and diuron on the microalgae Scenedesmus obliquus. Enzymatic activities were measured in the presence of these compounds alone and in mixture after a 24-h exposure to analyze the interactions between these herbicides. Catalase, ascorbate peroxidase, glutathione reductase, and glutathione S-transferase activities were investigated after the exposure of the microalgae to herbicide concentrations responsible for 25, 50, and 75% of growth inhibition. Antioxidative enzymes were significantly stimulated by oxyfluorfen, while no significant variation of catalase and ascorbate peroxidase activity was observed with diuron. However, this herbicide stimulated glutathione reductase and glutathione S-transferase of Scenedesmus obliquus. Growth inhibition test with oxyfluorfen and diuron in mixture showed an antagonistic interaction. At the enzyme level, antagonism was observed only with catalase. According to our data, diuron effect was not caused by an increased stimulation of algal antioxidative enzymes by the photosystem II herbicide.
A flow-cytometric method was developed and evaluated as a rapid ecotoxicological tool using cultures of the microalga Chlamydomonas reinhardtii (Chlorophyceae) under cadmium exposure. Three staining protocols were developed to assess the toxicological impact of this trace metal on algal physiology. Algal cells were exposed to total nominal cadmium concentrations of 5 and 100 µM. After 48 and 72 h exposure the fluorescent probes, fluorescein diacetate (FDA), dihydrorhodamine 123 (DHR123) and tetramethylrhodamine methyl ester (TMRM), were used to assess esterase activity, presence of reactive oxygen species and membrane potential, respectively. Results indicated that cell size, cell granularity and internal complexity were influenced by cadmium, confirming earlier findings on ultrastructural changes in microalgae exposed to trace metals. An increase was observed in the percentage of DHR123 positive cells as well as in their mean fluorescence intensity, on increasing cadmium concentration, confirming that this metal exerts its toxicity through the generation of reactive oxygen species. Furthermore, cadmium exposure resulted in an increase in esterase activity, as reflected in fluorescein fluorescence. We suggest this observation was linked to possible detoxification activity and defence mechanisms. Measurements of control samples during protocol optimization for TMRM proved not to be reproducible, leading us to defer any judgment on results of exposed samples and to conclude that TMRM does not seem suitable for flow cytometric use in algae. Our results demonstrate that although very rarely used in ecotoxicology, flow cytometry is a quick and convenient technique to assess toxic effects that can generate mechanistic information on the mode of action of contaminants.
The role of reactive oxygen species (ROS) in copper (Cu) toxicity to two freshwater green algal species, Pseudokirchneriella subcapitata (Korshikov) Hindák and Chlorella vulgaris Beij., was assessed to gain a better mechanistic understanding of this toxicity. Cu-induced formation of ROS was investigated in the two algal species and linked to short-term effects on photosynthetic activity and to long-term effects on cell growth. A light- and time-dependent increase in ROS concentrations was observed upon exposure to environmentally relevant Cu concentrations of 50 and 250 nM and was comparable in both algal species. However, effects of 250 nM Cu on photosynthesis were different, leading to a 12% reduction in photosynthetic activity in P. subcapitata, but not in C. vulgaris. These results indicate that differences in species-specific sensitivities measured as photosynthetic activity were not caused by differences in the cellular ROS content of the algae, but probably by different species-specific ROS defense systems. To investigate the role of ROS in Cu-mediated inhibition of photosynthesis, the ROS scavenger N-tert-butyl--phenylnitrone (BPN) was used, resulting in a reduction of Cu-induced ROS production up to control level and a complete restoration of photosynthetic activity of Cu-exposed P. subcapitata. This finding implied that ROS play a primary role in Cu toxicity to algae. Furthermore, we observed a time-dependent ROS release process across the plasma membrane. More than 90% of total ROS were determined to be extracellular in P. subcapitata, indicating an efficient method of cellular protection against oxidative stress.
Toxic effects of metals appear to be partly related to the production of reactive oxygen species (ROS), which can cause oxidative damage to cells. The ability of several redox active metals [Fe(III), Cu(II), Ag(I), Cr(III), Cr(VI)], nonredox active metals [Pb(II), Cd(II), Zn(II)], and the metalloid As(III) and As(V) to produce ROS at environmentally relevant metal concentrations was assessed. Cells of the freshwater alga Chlamydomonas reinhardtii P. A. Dang. were exposed to various metal concentrations for 2.5 h. Intracellular ROS accumulation was detected using an oxidation-sensitive reporter dye, 5-(and-6)-carboxy-2′,7′-dihydrodifluorofluorescein diacetate (H2DFFDA), and changes in the fluorescence signal were quantified by flow cytometry (FCM). In almost all cases, low concentrations of both redox and nonredox active metals enhanced intracellular ROS levels. The hierarchy of maximal ROS induction indicated by the increased number of stained cells compared to the control sample was as follows: Pb(II) > Fe(III) > Cd(II) > Ag(I) > Cu(II) > As(V) > Cr(VI) > Zn(II). As(III) and Cr(III) had no detectable effect. The effective free metal ion concentrations ranged from 10−6 to 10−9 M, except in the case of Fe(III), which was effective at 10−18 M. These metal concentrations did not affect algal photosynthesis. Therefore, a slightly enhanced ROS production is a general and early response to elevated, environmentally relevant metal concentrations.
Photosynthetic organisms constantly face the threat of photo-oxidative stress from fluctuating light conditions and environmental stress. Plants and algae have developed an array of defences to protect the chloroplast from reactive oxygen species. Genetic and physiological studies have shown that antioxidant responses are important to high-light acclimation, both by directly scavenging or quenching reactive oxygen intermediates and by contributing reducing power for alternative electron transport pathways and excess energy dissipation. At present, the signalling events leading to up-regulation of antioxidant defences in high light remain a mystery. Recent advances toward understanding acclimation to oxidative stress in both photosynthetic and non-photosynthetic model organisms may illuminate how plants and algae respond to high-light stress. Although the role of hydrogen peroxide in high-light acclimation has been investigated, less is known about responses to singlet oxygen, a form of reactive oxygen that poses a significant threat specifically to photosynthetic organisms. This review will discuss some intriguing new findings in that area, focusing on recent findings regarding the nature of singlet-oxygen responses in the chloroplast.
The green alga Chlamydomonas reinhardtii is a useful model of a photosynthetic cell. This unicellular eukaryote has been intensively used for studies of a number of physiological processes such as photosynthesis, respiration, nitrogen assimilation, flagella motility and basal body function. Its easy-to-manipulate and short life cycle make this organism a powerful tool for genetic analysis. Over the past 15 yr, a dramatically increased number of molecular technologies (including nuclear and organellar transformation systems, cosmid, yeast artificial chromosome (YAC) and bacterial artificial chromosome (BAC) libraries, reporter genes, RNA interference, DNA microarrays, etc.) have been applied to Chlamydomonas. Moreover, as parts of the Chlamydomonas genome project, molecular mapping, as well as whole genome and extended expressed sequence tag (EST) sequencing programs, are currently underway. These developments have allowed Chlamydomonas to become an extremely valuable model for molecular approaches to heavy metal homeostasis and tolerance in photosynthetic organisms.
When the fluorescence signal of a dye is being quantified, the staining protocol is an important factor in ensuring accuracy and reproducibility. Increasingly, lipophilic dyes are being used to quantify cellular lipids in microalgae. However, there is little discussion about the sensitivity of these dyes to staining conditions. To address this, microalgae were stained with either the lipophilic dyes often used for lipid quantification (Nile Red and BODIPY) or a lipophilic dye commonly used to stain neuronal cell membranes (DiO), and fluorescence was measured using flow cytometry. The concentration of the cells being stained was found not to affect the fluorescence. Conversely, the concentration of dye significantly affected the fluorescence intensity from either insufficient saturation of the cellular lipids or formation of dye precipitate. Precipitates of all three dyes were detected as events by flow cytometry and fluoresced at a similar intensity as the chlorophyll in the microalgae. Prevention of precipitate formation is, therefore, critical to ensure accurate fluorescence measurement with these dyes. It was also observed that the presence of organic solvents, such as acetone and dimethyl sulfoxide (DMSO), were not required to increase penetration of the dyes into cells and that the presence of these solvents resulted in increased cellular debris. Thus, staining conditions affected the fluorescence of all three lipophilic dyes, but Nile Red was found to have a stable fluorescence intensity that was unaffected by the broadest range of conditions and could be correlated to cellular lipid content.
The release of herbicides into the aquatic environment raises concerns about potential detrimental effects on ecologically important non-target species, such as unicellular algae, necessitating ecotoxicological risk assessment. Algal toxicity tests based on growth, a commonly assessed endpoint, are integrative, and hence do not provide information about underlying toxic mechanisms and effects. This limitation may be overcome by measuring more specific biochemical and physiological endpoints. In the present work, we developed and applied a novel multiple-endpoint assay, and analyzed the effects of the herbicides paraquat, diuron and norflurazon, each representing a specific mechanism of toxic action, on the single celled green alga Chlamydomonas reinhardtii. The endpoints added to assessment of growth were pigment content, maximum and effective photosystem II quantum yield, ATP content, esterase and oxidative activity. All parameters were measured at 2, 6 and 24h of exposure, except for growth and pigment content, which were determined after 6 and 24h only. Effective concentrations causing 50% of response (EC50s) and lowest observable effect concentrations (LOECs) were determined for all endpoints and exposure durations where possible. The assay provided a detailed picture of the concentration- and time-dependent development of effects elicited by the analyzed herbicides, thus improving the understanding of the underlying toxic mechanisms. Furthermore, the response patterns were unique to the respective herbicide and reflected the different mechanisms of toxicity. The comparison of the endpoint responses and sensitivities revealed that several physiological and biochemical parameters reacted earlier or stronger to disturbances than growth. Overall, the presented multiple-endpoint assay constitutes a promising basis for investigating stressor and toxicant effects in green algae.
Toxicities of ZnO, TiO2 and CuO nanoparticles to Pseudokirchneriella subcapitata were determined using OECD 201 algal growth inhibition test taking in account potential shading of light. The results showed that the shading effect by nanoparticles was negligible. ZnO nanoparticles were most toxic followed by nano CuO and nano TiO2. The toxicities of bulk and nano ZnO particles were both similar to that of ZnSO4 (72 h EC50 approximately 0.04 mg Zn/l). Thus, in this low concentration range the toxicity was attributed solely to solubilized Zn2+ ions. Bulk TiO2 (EC50=35.9 mg Ti/l) and bulk CuO (EC50=11.55 mg Cu/l) were less toxic than their nano formulations (EC50=5.83 mg Ti/l and 0.71 mg Cu/l). NOEC (no-observed-effect-concentrations) that may be used for risk assessment purposes for bulk and nano ZnO did not differ (approximately 0.02 mg Zn/l). NOEC for nano CuO was 0.42 mg Cu/l and for bulk CuO 8.03 mg Cu/l. For nano TiO2 the NOEC was 0.98 mg Ti/l and for bulk TiO2 10.1 mg Ti/l. Nano TiO2 formed characteristic aggregates entrapping algal cells that may contribute to the toxic effect of nano TiO2 to algae. At 72 h EC50 values of nano CuO and CuO, 25% of copper from nano CuO was bioavailable and only 0.18% of copper from bulk CuO. Thus, according to recombinant bacterial and yeast Cu-sensors, copper from nano CuO was 141-fold more bioavailable than from bulk CuO. Also, toxic effects of Cu oxides to algae were due to bioavailable copper ions. To our knowledge, this is one of the first systematic studies on effects of metal oxide nanoparticles on algal growth and the first describing toxic effects of nano CuO towards algae.
Flow cytometric methods for recognizing several groups of eukaryotic marine phytoplankton were tested using 26 laboratory cultures. Each culture was divided into three aliquots, and these samples were analyzed for (1) Coulter volume; (2) light scatter (magnitude and polarization properties of forward scattered light and magnitude of right-angle scattered light) and autofluorescence emission (phycoerythrin and chlorophyll); and (3) autofluorescence excitation (by 488 nm and 515 nm light).
Three kinds of cells could be easily distinguished from others in the culture collection: (1) The two cryptophytes and the rhodophyte had high phycoerythrin/chlorophyll ratios; (2) the two coccolithophores depolarized forward scattered light; and (3) the two pennate diatoms scattered only a relatively small amount of light in the forward direction compared with that at right angles. Mean chlorophyll fluorescence excited by blue light relative to that excited by green light was highest in the four chlorophytes, but there was overlap between some of these and some other kinds of cells. Unresolved cell types included centric diatoms, dinoflagellates, and naked coccolithophores. Forward light scatter and Coulter volume were closely related (except for the pennate diatoms) over a range of about 0.01 to 30 pL (equivalent spherical diameter about 3 to 40 μm), according to a logarithmic function.
Organic solvents can make their way into the environment as industrial wastes and components of pesticide formulations. In laboratory bioassays, the use of organic solvents is unavoidable since many pesticides and organic pollutants have low water solubilities and need to be dissolved in organic solvents prior to addition into experimental systems. So, one area of concern with laboratory bioassays is the stress imposed on test organisms by organic solvents. Most reports on the comparative toxicity of solvents towards test organisms deals with the effects of solvents on fish and aquatic invertebrates with some data available for blue-green algae and green algae. The US Environmental Protection Agency recommends maximum allowable limits of 0.05% solvent for acute tests and 0.01% for chronic tests but, in the literature, the nature of the solvent and the final concentration used vary among the different authors and are often higher than EPA limits due to problems associated with the use of small test volumes and toxicant solubility. Organic solvents can cause toxic effects on their own, but it has been also reported that they can interact with pesticides to alter toxicity. The first step in choosing a solvent for use in bioassays should be a detailed screening to identify solvents with inherently low toxicity to the test organism, followed by an interaction study (pesticide and solvent interactions) to choose the best concentration to use. The purpose of this study is to compare the inhibitory effects of our solvents used in pesticide bioassays towards the growth of two green algae. 18 refs., 4 figs., 1 tabs.
A ratio-fluorescence assay was developed for on-line localization and quantification of lipid oxidation in living cells. The assay explores the oxidative sensitivity of C11-BODIPY(581/591). Upon oxidation, the fluorescence of this fluorophore shifts from red to green. The probe incorporates readily into cellular membranes and is about twice as sensitive to oxidation as arachidonic acid. Using confocal microscopy, the cumene hydroperoxide-induced oxidation of C11-BODIPY(581/591) was visualized at the sub-cellular level in rat-1 fibroblasts. Preloading of the cells with tocopherol retarded this oxidation. The data demonstrate that C11-BODIPY(581/591) is a valuable tool to quantify lipid oxidation and anti-oxidant efficacy in single cells.
Cytotoxic effects of aquatic pollutants on microalgae are very heterogeneous, and they are influenced by environmental conditions and the test species. Stress produced by copper or paraquat addition to the culture medium of two microalgae was analysed by flow cytometry. Parameters assayed were: cell volume, chlorophyll a fluorescence and cell viability. The variety of results obtained in the present study reveals that flow cytometry is a useful tool in the toxicity tests with microalgae, both marine and freshwater species, and for different kind of pollutants.
Some herbicides act by binding to the exchangeable quinone site in the photosystem II (PSII) reaction centre, thus blocking electron transfer. In this article, it is hypothesized that the plant is killed by light-induced oxidative stress initiated by damage caused by formation of singlet oxygen in the reaction centre itself. This occurs when light-induced charge pairs in herbicide-inhibited PSII decay by a charge recombination route involving the formation of a chlorophyll triplet state that is able to activate oxygen. The binding of phenolic herbicides favours this pathway, thus increasing the efficiency of photodamage in this class of herbicides.
Flow cytometry is a rapid method for the quantitative measurement of light scattering and fluorescent properties of cells. Although this technique has been widely applied to biomedical and environmental studies, its potential as a tool in ecotoxicological studies has not yet been fully exploited. This article describes the application of flow cytometry to the development of bioassays with marine and freshwater algae for assessing the bioavailability of contaminants in waters and sediments.
C11-BODIPY(581/591) is a fluorescent radio-probe for indexing lipid peroxidation and antioxidant efficacy in model membrane systems and living cells, with excellent characteristics: (i) emission in the visible range of the electromagnetic spectrum, with good spectral separation of the nonoxidized (595 nm) and oxidized (520 nm) forms; (ii) has a high quantum yield and because of this, low labeling concentrations can be used, ensuring minimal perturbation of the membrane whilst retaining favorable signal to noise ratios; (iii) has a good photo-stability and displays very few fluorescence artifacts; (iv) is virtually insensitive to environmental changes, i.e., pH or solvent polarity; (v) is lipophilic and as such easily enters membranes; (vi) once oxidized, C11-BODIPY(581/591) remains lipophilic and does not spontaneously leave the lipid bilayer; (vii) C11-BODIPY(581/591) localizes in two distinct pools within the lipid bilayer, a shallow pool at 18 A and a deep pool at < 7.5 A from the center of the bilayer; (viii) is not cytotoxic to rat-1 fibroblasts up to 50 microM; (ix) is sensitive to a variety of oxy-radicals and peroxynitrite, but not to superoxide, nitric oxide, transition metal ions, and hydroperoxides per se; (x) its sensitivity to oxidation is comparable to that of endogenous fatty acyl moieties.
Interactions between photosynthesis, mitochondrial respiration (mitorespiration), and chlororespiration have been investigated in the green alga Chlamydomonas reinhardtii using flash illumination and a bare platinum electrode. Depending on the physiological status of algae, flash illumination was found to induce either a fast (t(1/2) approximately 300 ms) or slow (t(1/2) approximately 3 s) transient inhibition of oxygen uptake. Based on the effects of the mitorespiratory inhibitors myxothiazol and salicyl hydroxamic acid (SHAM), and of propyl gallate, an inhibitor of the chlororespiratory oxidase, we conclude that the fast transient is due to the flash-induced inhibition of chlororespiration and that the slow transient is due to the flash-induced inhibition of mitorespiration. By measuring blue-green fluorescence changes, related to the redox status of the pyridine nucleotide pool, and chlorophyll fluorescence, related to the redox status of plastoquinones (PQs) in C. reinhardtii wild type and in a photosystem I-deficient mutant, we show that interactions between photosynthesis and chlororespiration are favored when PQ and pyridine nucleotide pools are reduced, whereas interactions between photosynthesis and mitorespiration are favored at more oxidized states. We conclude that the plastid oxidase, similar to the mitochondrial alternative oxidase, becomes significantly engaged when the PQ pool becomes highly reduced, and thereby prevents its over-reduction.
Cyanobacteria must cope with the negative effects of ultraviolet B (280-315 nm) (UV-B) stress caused by their obligatory light requirement for photosynthesis. The adaptation of the cyanobacterium Anabaena sp. to moderate UV-B radiation has been observed after 2 weeks of irradiation, as indicated by decreased oxidative stress, decreased damage, recovered photosynthetic efficiency and increased survival. Oxidative stress in the form of UV-B-induced production of reactive oxygen species was measured in vivo with the oxidative stress-sensitive probe 2',7'-dichlorodihydrofluorescein diacetate. Photooxidative damage by UV-B radiation, including lipid peroxidation and DNA strand breakage, was determined by a modified method using thiobarbituric acid reactive substances and fluorometric analysis of DNA unwinding. Photosynthetic quantum yield was determined by pulse amplitude-modulated fluorometry. The results suggest that moderate UV-B radiation results in an evident oxidative stress, enhanced lipid peroxidation, increased DNA strand breaks, elevated chlorophyll bleaching as well as decreased photosynthetic efficiency and survival during the initial exposure. However, DNA strand breaks, photosynthetic parameters and chlorophyll bleaching returned to their unirradiated levels after 4-7 days of irradiation. Oxidative stress and lipid peroxidation appeared to respond later because decreases were observed after 7 days of radiation. The survival curve against irradiation time exhibited a close relationship with the changes in photosynthetic quantum yield and DNA damage, with little mortality after 4 days. Growth inhibition by UV-B radiation was observed during the first 7 days of radiation, whereas normal growth resumed even under UV-B stress thereafter. An efficient defense system was assumed to come into play to repair photosynthetic and DNA damage and induce the de novo synthesis of UV-sensitive proteins and lipids, allowing the organisms to adapt to UV-B stress successfully and survive as well as grow. No induction of mycosporine-like amino acids (MAA) was observed during the adaptation of Anabaena sp. to UV-B stress in our work. The adaptation of the cyanobacterium correlated with and could be caused by the oxidative stress and oxidative damage.
The susceptibility of the dinoflagellate Lingulodinium polyedrum to Arochlor 1254, a commercial mixture of polychlorinated biphenyl (PCB) congeners was examined through toxicity bioassays based on cell survival and measures of oxidative balance and adaptive response to PCB stress. The highest Arochlor 1254 concentration that did not cause observed effects (NOEC) on cell growth was 100 (48 h) and 25 ppb (96 h). The concentration that caused maximum effect (MEC) was 300 ppb (48 h) and 250 ppb (µl L–1) (96 h). The concentrations that promoted 50% cell death (LC50) were 146 and 122 ppb after 48 and 96 h, respectively. The cell numbers were lower after 96 h of exposure than after 48 h, suggesting that neither recovery nor growth occurred.
Under exposure to 120 ppb Arochlor 1254 for 48 h the oxidative damage in proteins was 121% higher than the control, as measured by reactive carbonyl levels, but no oxidative damage was found in lipids measured as malondialdehyde contents. Total superoxide dismutase (SOD) activity increased to plateau levels 146% greater than control values. The cells also exhibited increased ascorbate peroxidase (APx) activity (50%) and peridinin content (27%). No changes were observed in β-carotene under these experimental conditions. Therefore, SOD and APx induction and increased peridinin content may be principal primary adaptive responses to an increase of reactive oxygen species in Arochlor 1254 stress as indicated by protein oxidative damage and are an early marker of Arochlor 1254 contamination.
Heavy metals are environmental pollutants that have the potential to induce severe stress-reactions in organisms on land as well as in the sea. We have studied effects of short term sublethal concentrations of copper (Cu2+) and cadmium (Cd2+) on the reactive oxygen metabolism of the marine red macroalga Gracilaria tenuistipitata. Additions of either 0.2 ppm Cu2+ or 1 ppm Cd2+ caused decreased growth (approximately 60%), increased oxidation of lipids and increased oxidative damage to proteins as shown by increased content of protein carbonyl groups. Together this strongly suggests an induction of oxidative stress. Cu2+ caused more oxidative damage than Cd2+. As a response to the increased oxidative stress, addition of Cu2+ induced the activities of catalase, ascorbate peroxidase, and superoxide dismutase. In contrast, Cd2+ only caused increased catalase activity. Ten-fold lower concentrations of the metals did not cause an increase in enzyme activity. Both heavy metals also increased the content of the antioxidants beta-carotene and lutein. The results show that Cd2+ and, to a larger extent, Cu2+ induce oxidative stress in short-term experiments and the seaweed responds by increasing the activity of the reactive oxygen metabolism.
Copper-zinc superoxide dismutase (Cu,Zn-SOD) and manganese superoxide dismutase (Mn-SOD) in some model experiments in vitro demonstrated antioxidant as well as pro-oxidant properties. In the present study, yeast Saccharomyces cerevisiae lacking Mn-SOD were studied using Cu,Zn-SOD inhibitor N-N'-diethyldithiocarbamate (DDC) as a model system to study the physiological role of the yeast Cu,Zn-SOD. Yeast treatment by DDC caused dose-dependent inhibition of SOD in vivo, with 75% inhibition at 10mM DDC. The inhibition of SOD by DDC resulted in modification of carbonylprotein levels, indicated by a bell-shaped curve. The activity of glutathione reductase, isocitrate dehydrogenase, and glucose-6-phosphate dehydrogenase (enzymes associated with antioxidant) increased, demonstrating a compensatory effect in response to SOD inhibition by different concentrations of DDC. A strong positive correlation (R2=0.97) was found between SOD and catalase activities that may be explained by the protective role of SOD for catalase. All observed effects were absent in the isogenic SOD-deficient strain that excluded direct DDC influence. The results are discussed from the point of view that in vivo Cu,Zn-SOD of S. cerevisiae can demonstrate both anti- and pro-oxidant properties.
Endogenously produced pro-oxidant reactive species are essential to life, being involved in several biological functions. However, when overproduced (e.g. due to exogenous stimulation), or when the levels of antioxidants become severely depleted, these reactive species become highly harmful, causing oxidative stress through the oxidation of biomolecules, leading to cellular damage that may become irreversible and cause cell death. The scientific research in the field of reactive oxygen species (ROS) associated biological functions and/or deleterious effects is continuously requiring new sensitive and specific tools in order to enable a deeper insight on its action mechanisms. However, reactive species present some characteristics that make them difficult to detect, namely their very short lifetime and the variety of antioxidants existing in vivo, capable of capturing these reactive species. It is, therefore, essential to develop methodologies capable of overcoming this type of obstacles. Fluorescent probes are excellent sensors of ROS due to their high sensitivity, simplicity in data collection, and high spatial resolution in microscopic imaging techniques. Hence, the main goal of the present paper is to review the fluorescence methodologies that have been used for detecting ROS in biological and non-biological media.
This study was conducted to determine the role of algal-algal interactions in a multispecies microalgal population on their sensitivities to copper based on an enzyme inhibition assay using flow cytometric measures. Autofluorescence (chlorophyll a and phycocyanin) was used to identify species and count algal signals. The effect of multispecies population on copper toxicity of Microcystis aeruginousa was detected (1) at the same initial cell density, (2) at the same surface area, and (3) in the presence and absence of Chlorella pyrenoidosa and Scenedesmus obliquus. As copper concentrations increased, esterase activity of M. aeruginosa changed in a concentration-dependent manner. The 24 h EC(50) value of M. aeruginosa in the multispecies population was significantly (P < 0.05) higher than those in the single-species population. Compared with S. obliquus, the effect of C. pyrenoidosa on M. aeruginosa was more marked (the 24 h EC(50) value of copper on fluorescin diacetate fluorescence of M. aeruginosa was 11 microg/L). At 48 h copper exposure (6 microg/L) analysis of intracellular reactive oxygen species levels also showed similar algal-algal interactions in multispecies microalgal populations. The pigment assay suggested that these algal-algal interactions occurred only at low concentrations (< 13 microg/L, 24 and 48 h copper exposure). This study demonstrates the importance of using multispecies populations to estimate metal toxicity in natural waters.
Excessive copper concentrations, known to induce reactive oxygen species (ROS) formation, have been tested with respect to their effects on transcript abundance and related proteins involved in oxidative stress responses. The results show that the stromal photosynthetic functions were more ROS sensitive than the membrane-located reactions. The rbcL over-expression compensated for the damage only at 10 microM Cu, whereas the genetic stimulation of alpha-tocopherol biosynthesis led to the protection of membrane reactions up to 50-100 microM Cu. For this reason, the gradual growth drop of Chlamydomonas reinhardtii cultures observed under increasing Cu(2+) concentrations matched better with the loss of photosynthetic capacity than with those of photosynthetic quantum yields. According to Larcher's stress concept, the results allow the identification of gene markers for the alarm (rbcL), the hardening (FeSOD, VTE3) and the exhaustion [cyclin-dependent protein kinase (cdk), psbA] phases. These genes can be used to rapidly evaluate the state of oxidative stress in algae and putatively in other plant cells.
Part of the reduction in boar sperm motility and fertility associated with hypothermic liquid storage and cryopreservation may be due to membrane lipid peroxidation. Lipid peroxidation was monitored by the shift from red to green fluorescence emission of the lipophilic probe 4, 4-difluoro-5-(4-phenyl-1,3-butadienyl)-4-bora-3a,4a-diaza-s-indacene-3-undecanoic acid, C(11)BODIPY(581/591) (BODIPY), as measured by fluorescence-activated flow cytometry in live sperm (negative for propidium iodide). Experiments were conducted with Percoll-washed sperm to determine the specificity of BODIPY oxidation in the presence of different reactive oxygen species generators and metal chelators. Compared with no FeSO(4) and Na ascorbate, the combination of FeSO(4) and Na ascorbate (FeAc) increased (P < 0.01) the percentage of sperm containing oxidized BODIPY from <or=1 to >70% and increased (P < 0.05) BOD-IPY fluorescence intensity/cell by 5- to 10-fold after a 30-min incubation. Motility was depressed (P < 0.05) after exposure to FeAc, but viability was not affected. Of the reactive oxygen species generators tested, BODIPY oxidation was specific for FeAc, because menadione and H(2)O(2) had little or no effect. The oxidization of hydroethidine to ethidium was specific for menadione and H(2)O(2); FeAc had no effect. The presence of the metal chelators EDTA or deferoxamine mesylate at 3 and 9 muM inhibited FeAc-induced BODIPY oxidation and maintained motility. Experiments were conducted to determine the effect of liquid storage at 17 degrees C for 1 and 5 d and the effect of freeze-thawing on basal and FeAc-induced BODIPY oxidation. Basal BODIPY oxidation (no FeAc) was low in liquid stored and thawed viable sperm (1.3 and 3.4%, respectively). Although the incidence of basal or spontaneous membrane lipid peroxidation was low during liquid storage and after freeze-thawing, viable boar sperm were susceptible to FeAc-induced lipid peroxidation.
Ischemia-reperfusion injury induces cell death, but the responsible mechanisms are not understood. This study examined mitochondrial depolarization and cell death during ischemia and reperfusion. Contracting cardiomyocytes were subjected to 60-min ischemia followed by 3-h reperfusion. Mitochondrial membrane potential (DeltaPsi(m)) was assessed with tetramethylrhodamine methyl ester. During ischemia, DeltaPsi(m) decreased to 24 +/- 5.5% of baseline, but no recovery was evident during reperfusion. Cell death assessed by Sytox Green was minimal during ischemia but averaged 66 +/- 7% after 3-h reperfusion. Cyclosporin A, an inhibitor of mitochondrial permeability transition, was not protective. However, pharmacological antioxidants attenuated the fall in DeltaPsi(m) during ischemia and cell death after reperfusion and decreased lipid peroxidation as assessed with C11-BODIPY. Cell death was also attenuated when residual O(2) was scavenged from the perfusate, creating anoxic ischemia. These results suggested that reactive oxygen species (ROS) were important for the decrease in DeltaPsi(m) during ischemia. Finally, 143B-rho(0) osteosarcoma cells lacking a mitochondrial electron transport chain failed to demonstrate a depletion of DeltaPsi(m) during ischemia and were significantly protected against cell death during reperfusion. Collectively, these studies identify a central role for mitochondrial ROS generation during ischemia in the mitochondrial depolarization and subsequent cell death induced by ischemia and reperfusion in this model.
Glutathione (GSH) is the most abundant nonprotein thiol in eukaryotic cells and it protects cells by functioning as an antioxidant and a metal-binding ligand. Because glutathione readily undergoes oxidation-reduction reactions to combat oxidative stress, intracellular ratios of the reduced (GSH) to the oxidized (GSSG) forms of glutathione may serve as an important biomarker of exposure and effect of trace metals in eukaryotic cells. We compared sensitivity of glutathione ratios in the freshwater alga Chlamydomonas reinhardtii to the traditional endpoints of cell growth rates and chlorophyll a following exposure to Cu for periods of 6 and 24 h. A response of the GSH:GSSG ratio to Cu concentration was observed at Cu levels of 40 and 80 nM after exposure for both 6 and 24 h. The concentration of total GSH at 24 h was roughly half the value at 6 h after exposure to either 40 or 80 nM Cu. A response for cell growth rate was observed only at 24 h, whereby the average specific growth rate decreased from about 1.1 to 0.4 d(-1). The total Cu concentrations eliciting a cell response of 50%, effect concentrations (EC50s), after 24 h of exposure were similar (49.2, 49.8, and 38.2 nM Cu) and not significantly different for GSH:GSSG ratio, GSH levels, and specific growth, respectively. Total cell-associated Cu concentrations after exposure for 24 h were calculated from the EC50 endpoints and ranged from 13.3 to 17.0 fg/cell. Overall, thiol ratios were indicative of toxicity resulting from exposure to Cu, but precision may be greater for the cell growth rate endpoints.
The objective of the present investigation was to provide a diagnostic tool for the analysis of phytotoxic interactions between environmental contaminants and algae by application of flow cytometry. Therefore, an experimental design was developed consisting of synchronized Scenedesmus vacuolatus cell populations at defined cell-cycle stages, short-term exposure against different inhibitors with known molecular targets, and fluorochrome labeling of different metabolic processes. To discriminate cells with compromised metabolic processes from intact and metabolically inactive cells, references for every fluorochrome were defined using control and heat-treated populations. The experimental results showed that fluorescence markers are able to detect disturbance of specific cellular characteristics, such as membrane integrity, chlorophyll synthesis, and degradation. A differentiation of impacts on specific metabolic process caused by the reference inhibitors in concentration-dependent patterns could be seen using flow-cytometric fluorochrome analysis. These findings were compared with effects observed for N-phenyl-2-naphthylamine (PNA), a sediment contaminant of high phytotoxicity but unclear mode of action. Rhodamine 123 and cyano-ditolyl tetrazolium chloride detected significant metabolic changes for relevant exposures against PNA, thus pointing to compromised mitochondrial activity and changes in membrane potential as causes of phytotoxicity.
Developments in nanotechnology are leading to a rapid proliferation of new materials that are likely to become a source of engineered nanoparticles (ENPs) to the environment, where their possible ecotoxicological impacts remain unknown. The surface properties of ENPs are of essential importance for their aggregation behavior, and thus for their mobility in aquatic and terrestrial systems and for their interactions with algae, plants and, fungi. Interactions of ENPs with natural organic matter have to be considered as well, as those will alter the ENPs aggregation behavior in surface waters or in soils. Cells of plants, algae, and fungi possess cell walls that constitute a primary site for interaction and a barrier for the entrance of ENPs. Mechanisms allowing ENPs to pass through cell walls and membranes are as yet poorly understood. Inside cells, ENPs might directly provoke alterations of membranes and other cell structures and molecules, as well as protective mechanisms. Indirect effects of ENPs depend on their chemical and physical properties and may include physical restraints (clogging effects), solubilization of toxic ENP compounds, or production of reactive oxygen species. Many questions regarding the bioavailability of ENPs, their uptake by algae, plants, and fungi and the toxicity mechanisms remain to be elucidated.
C11-BODIPY581/ 591, an oxidation-sensitive fluorescent lipid peroxidation probe: (Micro)spectroscopic characterization and validation of methodology
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Drummen GPC, Van Liebergen LCM, Opden Kamp JAF, Post JA. C11-BODIPY581/ 591, an oxidation-sensitive fluorescent lipid peroxidation probe: (Micro)spectroscopic characterization and validation of methodology. Free Radic Biol Med 2002;33:473–490.
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