Unravelling joint cytotoxicity of ibuprofen and oxytetracycline on Chlamydomonas reinhardtii using a programmed cell death-related biomarkers panel.

Pharmaceutical active compounds (PhACs) are emerging contaminants that pose a growing concern due to their ubiquitous presence and harmful impact on aquatic ecosystems. Among PhACs, the anti-inflammatory ibuprofen (IBU) and the antibiotic oxytetracycline (OTC) are two of the most used compounds whose presence has been reported in different aquatic environments worldwide. However, there is still scarce information about the cellular and molecular alterations provoked by IBU and OTC on aquatic photosynthetic microorganisms as microalgae, even more if we refer to their potential combined toxicity. To test the cyto- and genotoxicity provoked by IBU, OTC and their binary combination on Chlamydomonas reinhardtii, a flow cytometric panel was performed after 24 h of single and co-exposure to both contaminants. Assayed parameters were cell vitality, metabolic activity, intracellular ROS levels, and other programmed cell death (PCD)-related biomarkers as cytoplasmic and mitochondrial membrane potentials and caspase-like and endonuclease activities. In addition, a nuclear DNA fragmentation analysis by comet assay was carried out. For most of the parameters analysed (vitality, metabolic activity, cytoplasmic and mitochondrial membrane potentials, and DNA fragmentation) the most severe damages were observed in the cultures exposed to the binary mixture (IBU+OTC), showing a joint cyto- and genotoxicity effect. Both PhACs and their mixture caused a remarkable decrease in cell proliferation and metabolic activity and markedly increased intracellular ROS levels, parallel to a noticeable depolarization of cytoplasmic and mitochondrial membranes. Moreover, a strong increase in both caspase and endonuclease activities as well as a PCD-related loss of nuclear DNA integrity was observed in all treatments. Results analysis showed that the PhACs caused cell death on this non-target organism, involving mitochondrial membrane depolarization, enhanced ROS production and activation of PCD process. Thus, PCD should be an applicable toxicological target for unraveling the harmful effects of co-exposure to PhACs in aquatic organisms as microalgae.

Cytotoxicity of BP-3 and BP-4: Blockage of extrusion pumps, oxidative damage and programmed cell death on Chlamydomonas reinhardtii.

The health concern associated with the dangers related to exposure to UV radiation has led to an increase in the use of sunscreens containing UV-filters that can reach aquatic environments and possibly affect ecosystems. Benzophenone-3 (BP-3) and benzophenone-4 (BP-4) are two of the most used UV-filters. In the present work, the microalga Chlamydomonas reinhardtii was exposed to several concentrations of both chemicals. To evaluate their potential cytotoxicity on microalgal cells, different parameters were analysed including fast response biomarkers (increase in intracellular free Ca2+) as well as biomarkers related with the presence of oxidative stress (lipid peroxidation), energy metabolism (photosynthetic yield and cytoplasmic lipid accumulations), cell division (proliferation and F-actin content), programmed cell death (PCD) (caspase activation and DNA fragmentation) and possible mechanisms of resistance to xenobiotics (operation of extrusion pumps and presence of autophagic vacuoles). Results showed an increment of the percentage of cells with cytosolic free Ca2+ that could act as a secondary messenger in response to the stress. A decrease in photosynthetic yield and an increase in cytoplasmic lipid accumulations and lipid peroxidation levels were also detected. In addition, a decrease in cell proliferation was observed, linked to a decrease in the percentage of cells with F-actin. The increase observed in the microalgal population with caspase activity, together with the DNA fragmentation and the alterations in the cytoskeleton, suggested the induction of processes linked to PCD. Moreover, a blockage of extrusion pumps, which could be related to the toxicity mechanism of these compounds, and an increase in autophagic vacuoles, as an attempt to repair the damage caused by benzophenones, were detected. Overall, these biomarkers indicate that both UV-filters can be a serious threat to non-target photosynthetic microorganisms in aquatic environments, although BP-3 affected C. reinhardtii more markedly.

Differential toxicity of the UV-filters BP-3 and BP-4 in Chlamydomonas reinhardtii: A flow cytometric approach.

Due to the concern about the negative effects of exposure to sunlight, UV-filters are being introduced in all kind of cosmetic formulas. Wastewater treatment plants are not able to remove and/or degrade them; consequently they find their way into rivers, lakes and oceans. These chemicals are acquiring a concerning status due to their increasingly common use and the potential risk for the environment. Benzophenone-3 (BP-3) and Benzophenone-4 (BP-4) are broad-spectrum UV-filters used for the same purpose in personal care products, insecticides and plastic bags; however, after 96 h of exposure to several concentrations of these UV-filters, the growth of C. reinhardtii was more affected by BP-3 than by BP-4, being the 96 h-EC50 for growth 5 mg L-1 and 38 mg L-1, respectively. Based on these values Chlamydomonas reinhardtii cultures were exposed during 24 h to 2.5, 5 and 10 mg L-1 of BP-3 and 19, 38 and 76 mg L-1 of BP-4. A cytometric panel was carried out to evaluate the effect of sublethal concentrations of these UV-filters, thus several cytotoxicity biomarkers were analysed, including chlorophyll a fluorescence, viability, metabolic activity, oxidative stress, cytoplasmic and mitochondrial membrane potentials, and intracellular pH. BP-3 and BP-4 affect C.reinhardtii cells in a different way, showing differences for three of the examined parameters. Chlorophyll a fluorescence and mitochondrial membrane potential showed a significant increase (p < 0.05) in BP-3 and a significant decrease in BP-4, whereas viability only decreased significantly in the highest concentrations of BP-3. Regarding to the other parameters analysed, a similar pattern of cytotoxicity was observed. Growth rate, vital population and metabolic activity (esterase activity) and intracellular pH decreased significantly and cytoplasmic membrane potential and ROS levels increased significantly in cultures exposed to both pollutants.

Does a short-term exposure to atrazine provoke cellular senescence in Chlamydomonas reinhardtii?

The aim of this study was to investigate the impacts and modes of action of a chemical and nutrient deprivation on the cellular senescence process of Chlamydomonas reinhardtii. Several molecular and cellular parameters related to senescence phenomena were monitored in C. reinhardtii cells exposed for 24h to a sublethal concentration (0.25µM) of the herbicide atrazine and in unexposed 96h cells in an early stationary phase of growth. All endpoints showed the same pattern of response between treatments, except for the intracellular level of calcium, where a significant increase was observed in 24h-exposed cultures compared to 24h-log controls. Results also indicated that cell viability remained above 98% for all conditions. Reactive oxygen species (ROS) levels and caspase activity increased in all experimental cultures with respect to 24h-log controls and alterations in the nuclear morphology and cells with auto-phagosomes were observed in all treatments. However, a decrease in lipid peroxidation was detected which could be related to the observed increment of autophagic vacuoles that recycle damaged material, such as altered lipids in microalgal membranes. Furthermore, responses at the molecular level were also investigated. Gene transcription analyses, carried out by RT-qPCR technique, indicated an increase in transcripts for genes encoding glutathione S-transferase (GST) and ascorbate peroxidase (APX I) and a decrease for those encoding catalase (CAT), glutathione peroxidase (GPX) and Mn-superoxide dismutase (SOD-1) in both experimental treatments. Overall molecular and cellular results suggest that a short-term exposure to a sublethal concentration of atrazine may induce senescence features in microalgal cells which are the base of aquatic food webs.

Calcium mediates the cellular response of Chlamydomonas reinhardtii to the emerging aquatic pollutant Triclosan.

The present study was aimed at investigating the role of intracellular free calcium, [Ca2+]c, in the early cellular response of the green alga Chlamydomonas reinhardtii to the emergent pollutant Triclosan (13.8µM; 24h of exposure). There is a growing concern about the persistence and toxicity of this antimicrobial in aquatic environments, where non-target organisms such as C. reinhardtii, a primary producer of ecological relevance, might be severely impacted. A mechanistic study was undertaken which combined flow cytometry protocols, physiological as well as gene expression analysis. As an early response, Triclosan strongly altered [Ca2+]c homeostasis which could be prevented by prechelation with the intracellular calcium chelator BAPTA-AM. Triclosan induced ROS overproduction which ultimately leads to oxidative stress with loss of membrane integrity, membrane depolarization, photosynthesis inhibition and mitochondrial membrane depolarization; within this context, Triclosan also induced an increase in caspase 3/7 activity and altered the expression of metacaspase genes which are indicative of apoptosis. All these adverse outcomes were dependent on [Ca2+]c. Interestingly, an interconnection between [Ca2+]c alterations and increased ROS formation by Triclosan was found. Taken altogether these results shed light on the mechanisms behind Triclosan toxicity in the green alga Chlamydomonas reinhardtii and demonstrate the role of [Ca2+]c in mediating the observed toxicity.

Flow cytometric assay to assess short-term effects of personal care products on the marine microalga Tetraselmis suecica.

Large quantities of personal care products (PCPs) are used daily and many of their chemical ingredients are subsequently released into marine environments. Cultures of the marine microalga Tetraselmis suecica were exposed for 24 h to three emerging compounds included in the main classes of PCPs: the UV filter benzophenone-3 (BP-3), the disinfectant triclosan (TCS) and the fragrance tonalide (AHTN). Concentrations tested, expressed as cellular quota (pg cell-1), ranged from 5 to 40 for BP-3, from 2 to 16 for TCS and from 1.2 to 2.4 for AHTN. A small cytometric panel was carried out to evaluate key cytotoxicity biomarkers including inherent cell properties, growth and metabolic activity and cytoplasmic membrane properties. BP-3 caused a significant increase in growth rate, metabolic activity and chlorophyll a fluorescence from 10 pg cell-1. However, growth and esterase activity decreased in cells exposed to all TCS and AHTN concentrations, except the lowest ones. Also these two compounds provoked a significant swelling of cells, more pronounced in the case of TCS-exposed cells. Although all treated cells remained viable, changes in membrane potential were observed. BP-3 and AHTN caused a significant depolarization of cells from 10 to 1.6 pg cell-1, respectively; however all TCS concentrations assayed caused a noticeable hyperpolarization of cells. Metabolic activity and cytoplasmic membrane potential were the most sensitive parameters. It can be concluded that the toxicological model used and the toxicological parameters evaluated are suitable to assess the toxicity of these emerging contaminants.

Early alterations on photosynthesis-related parameters in Chlamydomonas reinhardtii cells exposed to atrazine: A multiple approach study.

Chlamydomonas reinhardtii cells were exposed to a sublethal concentration of the widespread herbicide atrazine for 3h. Physiological cellular parameters, such as chlorophyll a fluorescence and oxidative stress monitored by flow cytometry and pigments levels were altered in microalgal cells exposed to 0.25 µM of atrazine. Furthermore, the effects of this herbicide on C. reinhardtii were explored using "omics" techniques. Transcriptomic analyses, carried out by RNA-Seq technique, displayed 9 differentially expressed genes, related to photosynthesis, between control cultures and atrazine exposed cultures. Proteomic profiles were obtained using iTRAQ tags and MALDI-MS/MS analysis, identifying important changes in the proteome during atrazine stress; 5 proteins related to photosynthesis were downexpressed. The results of these experiments advance the understanding of photosynthetic adjustments that occur during an early herbicide exposure. Inhibition of photosynthesis induced by atrazine toxicity will affect the entire physiological and biochemical states of microalgal cells.

Acute effects of a prooxidant herbicide on the microalga Chlamydomonas reinhardtii: Screening cytotoxicity and genotoxicity endpoints.

Since recent evidence has demonstrated that many types of chemicals exhibit oxidative and/or genotoxic potential on living organisms, reactive oxygen species (ROS) formation and DNA damage are currently the best accepted paradigms to assess the potential hazardous biological effects of a wide range of contaminants. The goal of this study was to evaluate the sensitivity of different cytotoxicity and genotoxicity responses on the model microalga Chlamydomonas reinhardtii exposed to the prooxidant herbicide paraquat. In addition to the growth endpoint, cell viability, mitochondrial membrane potential and presence of reactive oxygen species (ROS) were assayed as potential markers of cytotoxicity using flow cytometry (FCM). To study the effects of paraquat on C. reinhardtii DNA, several genotoxicity approaches were implemented for the first time in an ecotoxicological study on microalgae. Oxidative DNA base damage was analysed by measuring the oxidative DNA lesion 8-OHdG by FCM. DNA fragmentation was analysed by different methods: comet assay, and cell cycle analysis by FCM, with a particular focus on the presence of subG1-nuclei. Finally, effects on morphology of nuclei were monitored through DAPI staining. The evaluation of these endpoints showed that several physiological and biochemical parameters reacted to oxidative stress disturbances with greater sensitivity than integrative parameters such as growth rates or cell viability. The experiments revealed concentration-dependent cytotoxicity (ROS formation, depolarization of mitochondrial membrane), genotoxicity (oxidative DNA damage, DNA strand breakage, alterations in nuclear morphology), and cell cycle disturbances (subG1-nuclei, decrease of 4N population) in paraquat-treated cells. Overall, the genotoxicity results indicate that the production of ROS caused by exposure to paraquat induces oxidative DNA damage followed by DNA single- and double-strand breaks and cell cycle alterations, possibly leading to apoptosis in C. reinhardtii cells. This is supported by the observation of typical hallmarks of apoptosis, such as mitochondrial membrane depolarization, alterations in nuclear morphology and subG1 nuclei in cells exposed to the highest assayed concentrations. To our knowledge, this is the first study that provides a comprehensive analysis of oxidative DNA base damage in unicellular algal cells exposed to a prooxidant pollutant, as well as of its possible relation with other physiological effects. These results reinforce the need for additional studies on the genotoxicity of environmental pollutants on ecologically relevant organisms such as microalgae that can provide a promising basis for the characterization of potential pollutant hazards in the aquatic environment.

Chlamydomonas reinhardtii cells adjust the metabolism to maintain viability in response to atrazine stress.

Chlamydomonas reinhardtii cells were exposed to a sublethal concentration of the widespread herbicide atrazine for 3 and 24h. Physiological parameters related to cellular energy status, such as cellular activity and mitochondrial and cytoplasmic membrane potentials, monitored by flow cytometry, were altered in microalgal cells exposed to 0.25µM of atrazine. Transcriptomic analyses, carried out by RNA-Seq technique, displayed 12 differentially expressed genes between control cultures and atrazine-exposed cultures at both tested times. Many cellular processes were affected, but the most significant changes were observed in genes implicated in amino acid catabolism and respiratory cellular process. Obtained results suggest that photosynthesis inhibition by atrazine leads cells to get energy through a heterotrophic metabolism to maintain their viability.

Suitability of cytotoxicity endpoints and test microalgal species to disclose the toxic effect of common aquatic pollutants.

Pulse discharges of chemicals to aquatic environments may lead to high concentrations of them in surface waters for short periods of time, but enough to induce toxic effects on aquatic organisms; however, no many methods allow an early warning of toxicity of these agents. Acute effects of one representative chemical from each of three of the main groups of aquatic pollutants (pesticides, metals and pharmaceuticals) are studied on two green microalgal species (Chlamydomonas moewusii and Chlorella vulgaris). Flow cytometry protocols were used to detect the potential application of chlorophyll a fluorescent emission, cell viability, metabolic activity and membrane potential as cytotoxicity endpoints, besides an epifluorescence microscopy protocol for comet assay to detect genotoxicity level of cells. Obtained results confirm the suitability of them for the prospective assessment of the potential cytotoxicity of these aquatic pollutants. The two microalgal species analysed could be used as indicators in toxicity bioassays, being C. moewusii more sensitive than C. vulgaris. Among cell parameters assayed, the metabolic activity and the primary DNA damage stood out as sensitive cytotoxicity endpoints.

Toxicity induced by three antibiotics commonly used in aquaculture on the marine microalga Tetraselmis suecica (Kylin) Butch.

Aquaculture facilities are a potential source of antibiotics to the aquatic ecosystems. The presence of these compounds in the environment may have deleterious effects on non-target aquatic organisms such as microalgae, which are often used as biological indicators of pollution. Therefore, the toxicity induced by chloramphenicol (CHL), florphenicol (FLO) and oxytetracycline (OTC), three antibiotics widely used in aquaculture, on the marine microalga Tetraselmis suecica was evaluated. Growth inhibition and physiological and biochemical parameters were analysed. All three antibiotics inhibited growth of T. suecica with 96 h IC50 values of 11.16, 9.03 and 17.25 mg L(-1) for CHL, FLO and OTC, respectively. After 24 h of exposure no effects on growth were observed and cell viability was also unaffected, whereas a decrease in esterase activity, related with cell vitality, was observed at the higher concentrations assayed. Photosynthesis related parameters such as chlorophyll a cellular content and autofluorescence were also altered after 24 h of antibiotics addition. It can be concluded that T. suecica was sensitive to the three antibiotics tested.

Screening acute cytotoxicity biomarkers using a microalga as test organism.

The present study checked the suitability of the integration of flow cytometry (FCM) as technique and a freshwater microalga (Chlamydomonas moewusii) as cell system model for ecotoxicological studies, looking for sensitive biomarkers of acute cytotoxicity of potential contaminants in aquatic systems. The detection of the potential acute toxicity of a pollutant is of interest because pulse discharges of contaminants to natural waters could lead to high concentrations of these substances that are only present for short periods of time but can affect aquatic organisms such as microalgae. Physiological alterations in C. moewusii cells were analysed after 1h of exposure to different concentrations of the herbicide paraquat. Cell viability was not affected, but the acute toxicity of paraquat was evident at other levels of cell physiology. Herbicide-treated cells showed lower autofluorescence and higher size and internal complexity, lower esterase activity and lower mitochondrial membrane potential. Paraquat induced the depolarisation of the plasma membrane and the increase of intracellular free calcium level and cytosolic pH in a concentration-dependent percentage of cells. All these effects can be related to the oxidative stress induced by the herbicide, as revealed the significantly increased intracellular levels of reactive oxygen species in cultures exposed to paraquat concentrations which induced the physiological alterations mentioned above. Excluding cell viability and mitochondrial membrane potential, these cytotoxicity endpoints could be considered sensitive biomarkers for the short-term exposure to pollutants such as herbicides.

Flow cytometric analysis to evaluate physiological alterations in herbicide-exposed Chlamydomonas moewusii cells.

Investigation of herbicide toxicology in non-target aquatic primary producers such as microalgae is of great importance from an ecological point of view. In order to study the toxicity of the widely used herbicide paraquat on freshwater green microalga Chlamydomonas moewusii, physiological changes associated with 96 h-exposures to this pollutant were monitored using flow cytometry (FCM) technique. Intracellular reactive oxygen species concentration, cytoplasmic membrane potential, metabolic activity and cell protein content were monitored to evaluate the toxicological impact of paraquat on algal physiology. Results showed that herbicide paraquat induced oxidative stress in C. moewusii cells, as it indicated the increase of both superoxide anion and hydrogen peroxide levels observed in non-chlorotic cells of cultures exposed to increasing herbicide concentrations. Furthermore, a progressive increase in the percentage of depolarised cells and a decrease in the metabolic activity level were observed in response to paraquat when non-chlorotic cells were analysed. Chlorotic cells were probably non-viable cells, based on the cytoplasmic membrane depolarisation, its metabolically non-active state and its drastically reduced protein content. In view of the obtained results, we have concluded that a range of significant physiological alterations, detected by flow cytometry, occur when C. moewusii, an ubiquitous microalga in freshwater environments, is challenged with environmentally relevant concentrations of the herbicide paraquat.

Characterization of cell response in Chlamydomonas moewusii cultures exposed to the herbicide paraquat: Induction of chlorosis.

The use of herbicides constitutes the principal method of weed control, but the introduction of these compounds into the aquatic environment can provoke severe consequences for non-target organisms such as microalgae. Effects of the widely used herbicide paraquat were assessed on the green freshwater microalga Chlamydomonas moewusii by means of the analysis of its photosynthetic pigment content, using a traditional spectrophotometric technique that provides population bulk measurements, and by means of flow cytometry, which allowed characterizing the microalgal response at a single-cell level. Results obtained reveal that paraquat concentrations above 50nM induce chlorosis in a percentage of microalgal cells depending on herbicide concentration and exposure time, as reflected by a reduced cell chlorophyll autofluorescence and pigment content of the biomass. Cell viability in these cultures was also reduced in a concentration dependent way. The possibility of analysing chlorotic and non-chlorotic subpopulations separately allowed the study of morphological properties and physiological status of both cell types, leading to the conclusion that chlorotic cells are non-viable cells, based on their reduced size and complexity and their inability to be stained in the fluorescein diacetate assay. In the case of non-chlorotic cells, cell viability was reduced with the increase of paraquat concentration. Non-chlorotic cells in these cultures showed an increased size and complexity in comparison with control cells, probably due to a growth inhibition. Chlorophyll fluorescence was the most sensitive parameter since even cells exposed to the lowest concentration assayed, 50nM, although not chlorotic, showed a significantly reduced chlorophyll fluorescence with respect to control cells, reflected also by a reduced chlorophyll content of the biomass.

Cell proliferation alterations in Chlorella cells under stress conditions.

Very little is known about growth and proliferation in relation to the cell cycle regulation of algae. The lack of knowledge is even greater when referring to the potential toxic effects of pollutants on microalgal cell division. To assess the effect of terbutryn, a triazine herbicide, on the proliferation of the freshwater microalga Chlorella vulgaris three flow cytometric approaches were used: (1) in vivo cell division using 5-,6-carboxyfluorescein diacetate succinimidyl ester (CFSE) staining was measured, (2) the growth kinetics were determined by cytometric cell counting and (3) cell viability was evaluated with the membrane-impermeable double-stranded nucleic acid stain propidium iodide (PI). The results obtained in the growth kinetics study using CFSE to identify the microalgal cell progeny were consistent with those determined by cytometric cell counting. In all C. vulgaris cultures, each mother cell had undergone only one round of division through the 96 h of assay and the cell division occurred during the dark period. Cell division of the cultures exposed to the herbicide was asynchronous. Terbutryn altered the normal number of daughter cells (4 autospores) obtained from each mother cell. The number was only two in the cultures treated with 250 nM. The duration of the lag phase after the exposure to terbutryn could be dependent on the existence of a critical cell size to activate cytoplasmic division. Cell size, complexity and fluorescence of chlorophyll a of the microalgal cells presented a marked light/dark (day/night) cycle, except in the non-dividing 500 nM cultures, where terbutryn arrested cell division at the beginning of the cycle. Viability results showed that terbutryn has an algastatic effect in C. vulgaris cells at this concentration. The rapid and precise determination of cell proliferation by CFSE staining has allowed us to develop a model for assessing both the cell cycle of C. vulgaris and the in vivo effects of pollutants on growth and reproduction at microalgal cell level.

Atrazine-induced chlorosis in Synechococcus elongatus cells.

The effects of a widely used herbicide, atrazine, on the freshwater cyanobacterium Synechococcus elongatus were studied. The cyanobacteria were exposed to varying concentrations of atrazine (0.025, 0.05, 0.1, 0.25, and 0.75 microM) for 96 h. Different parameters such as growth, autofluorescence of chlorophyll a, pigment content, volume, and internal granularity of the cells were determined daily. Differences were detected between cultures with and cultures without atrazine for the parameters analyzed. Atrazine exposure induced the process of chlorosis in cyanobacterial cells, given that this herbicide has an effect on photosynthesis, chlorotic subpopulations having low values of chlorophyll a autofluorescence. More unpigmented subpopulations (chlorotic) appeared as the atrazine concentration increased and better growth rates resulted. Atrazine also induced changes in cell volume and internal granularity, these being most apparent after 48 h of exposure and in cultures with higher atrazine concentrations (0.25 and 0.75 microM).