Mitochondria dysfunction is one of the causes of diclofenac toxicity in the green alga Chlamydomonas reinhardtii.

Background: Non-steroidal anti-inflammatory drugs (NSAIDs), such as diclofenac (DCF), form a significant group of environmental contaminants. When the toxic effects of DCF on plants are analyzed, authors often focus on photosynthesis, while mitochondrial respiration is usually overlooked. Therefore, an in vivo investigation of plant mitochondria functioning under DCF treatment is needed. In the present work, we decided to use the green alga Chlamydomonas reinhardtii as a model organism. Methods: Synchronous cultures of Chlamydomonas reinhardtii strain CC-1690 were treated with DCF at a concentration of 135.5 mg × L-1, corresponding to the toxicological value EC50/24. To assess the effects of short-term exposure to DCF on mitochondrial activity, oxygen consumption rate, mitochondrial membrane potential (MMP) and mitochondrial reactive oxygen species (mtROS) production were analyzed. To inhibit cytochrome c oxidase or alternative oxidase activity, potassium cyanide (KCN) or salicylhydroxamic acid (SHAM) were used, respectively. Moreover, the cell's structure organization was analyzed using confocal microscopy and transmission electron microscopy. Results: The results indicate that short-term exposure to DCF leads to an increase in oxygen consumption rate, accompanied by low MMP and reduced mtROS production by the cells in the treated populations as compared to control ones. These observations suggest an uncoupling of oxidative phosphorylation due to the disruption of mitochondrial membranes, which is consistent with the malformations in mitochondrial structures observed in electron micrographs, such as elongation, irregular forms, and degraded cristae, potentially indicating mitochondrial swelling or hyper-fission. The assumption about non-specific DCF action is further supported by comparing mitochondrial parameters in DCF-treated cells to the same parameters in cells treated with selective respiratory inhibitors: no similarities were found between the experimental variants. Conclusions: The results obtained in this work suggest that DCF strongly affects cells that experience mild metabolic or developmental disorders, not revealed under control conditions, while more vital cells are affected only slightly, as it was already indicated in literature. In the cells suffering from DCF treatment, the drug influence on mitochondria functioning in a non-specific way, destroying the structure of mitochondrial membranes. This primary effect probably led to the mitochondrial inner membrane permeability transition and the uncoupling of oxidative phosphorylation. It can be assumed that mitochondrial dysfunction is an important factor in DCF phytotoxicity. Because studies of the effects of NSAIDs on the functioning of plant mitochondria are relatively scarce, the present work is an important contribution to the elucidation of the mechanism of NSAID toxicity toward non-target plant organisms.

Diclofenac Interacts with Photosynthetic Apparatus: Isolated Spinach Chloroplasts and Thylakoids as a Model System.

Diclofenac, often detected in environmental samples, poses a potential hazard to the aquatic environment. The present study aimed to understand the effect of this drug on photosynthetic apparatus, which is a little-known aspect of its phytotoxicity. Chloroplasts and thylakoids isolated from spinach (Spinacia oleracea) were used for this study and treated with various concentrations of diclofenac (from 125 to 4000 µM). The parameters of chlorophyll a fluorescence (the OJIP test) as measurements for both the intact chloroplasts and the thylakoid membranes revealed that isolated thylakoids showed greater sensitivity to the drug than chloroplasts. The relatively high concentration of diclofenac that is required to inhibit chloroplast and thylakoid functions suggests a narcotic effect of that drug on photosynthetic membranes, rather than a specific interaction with a particular element of the electron transport chain. Using confocal microscopy, we confirmed the degradation of the chloroplast structure after DCF treatment, which has not been previously reported in the literature. In conclusion, it can be assumed that diclofenac's action originated from a non-specific interaction with photosynthetic membranes, leading to the disruption in the function of the electron transport chain. This, in turn, decreases the efficiency of photosynthesis, transforming part of the PSII reaction centers into heat sinks and enhancing non-photochemical energy dissipation.

Individual architecture and photosynthetic performance of the submerged form of Drosera intermedia Hayne.

Drosera intermedia grows in acidic bogs in parts of valleys that are flooded in winter, and that often dry out in summer. It is also described as the sundew of the most heavily hydrated habitats in peatlands, and it is often found in water and even underwater. This sundew is the only one that can tolerate long periods of submersion, and more importantly produces a typical submerged form that can live in such conditions for many years. Submerged habitats are occupied by D. intermedia relatively frequently. The aim of the study was to determine the environmental conditions and architecture of individuals in the submerged form of D. intermedia. The features of the morphological and anatomical structure and chlorophyll a fluorescence of this form that were measured were compared with analogous ones in individuals that occurred in emerged and peatland habitats. The submerged form occurred to a depth of 20 cm. Compared to the other forms, its habitat had the highest pH (4.71-4.92; Me = 4.71), the highest temperature and substrate hydration, and above all, the lowest photosynthetically active radiation (PAR; 20.4-59.4%). This form differed from the other forms in almost all of the features of the plant's architecture. It is particularly noteworthy that it had the largest main axis height among all of the forms, which exceeded 18 cm. The number of living leaves in a rosette was notable (18.1 ± 8.1), while the number of dead leaves was very low (6.9 ± 3.8). The most significant differences were in the shape of its submerged leaves, in which the length of the leaf blade was the lowest of all of the forms (0.493 ± 0.15 mm; p < 0.001) and usually the widest. The stem cross-sectional area was noticeably smaller in the submerged form than in the other forms, the xylem was less developed and collaterally closed vascular bundles occurred. Our analysis of the parameters of chlorophyll fluorescence in vivo revealed that the maximum quantum yield of the primary photochemistry of photosystem II is the highest for the submerged form (Me = 0.681), the same as the maximum quantum yield of the electron transport (Me φE0 = 0.183). The efficiency of energy use per one active reaction center of photosystem II (RC) was the lowest in the submerged form (Me = 2.978), same as the fraction of energy trapped by one active RC (Me = 1.976) and the non-photochemical energy dissipation (DI0/RC; Me = 0.916). The ET0/RC parameter, associated with the efficiency of the energy utilization for electron transport by one RC, in the submerged plant reached the highest value (Me = 0.489). The submerged form of D. intermedia clearly differed from the emerged and peatland forms in its plant architecture. The submerged plants had a thinner leaf blade and less developed xylem than the other forms, however, their stems were much longer. The relatively high photosynthetic efficiency of the submerged forms suggests that most of the trapped energy is utilized to drive photosynthesis with a minimum energy loss, which may be a mechanism to compensate for the relatively small size of the leaf blade.

Multi-biomarker response of cyanobacteria Synechocystis salina and Microcystis aeruginosa to diclofenac.

The cyanobacterial response to pharmaceuticals is less frequently investigated compared to green algae. Pharmaceuticals can influence not only the growth rate of cyanobacteria culture, but can also cause changes at the cellular level. The effect of diclofenac (DCF) as one of the for cyanobacteria has been rarely tested, and DCF has never been applied with cellular biomarkers. The aim of this work was to test the response of two unicellular cyanobacteria (Synechocystis salina and Microcystis aeruginosa) toward DCF (100 mg L-1) under photoautotrophic growth conditions. Such endpoints were analyzed as cells number, DCF uptake, the change in concentrations of photosynthetic pigments, the production of toxins, and chlorophyll a in vivo fluorescence. It was noted that during a 96 h exposure, cell proliferation was not impacted. Nevertheless, a biochemical response was observed. The increased production of microcystin was noted for M. aeruginosa. Due to the negligible absorption of DCF into cells, it is possible that the biochemical changes are induced by an external signal. The application of non-standard biomarkers demonstrates the effect of DCF on microorganism metabolism without a corresponding effect on biomass. The high resistance of cyanobacteria to DCF and the stimulating effect of DCF on the secretion of toxins raise concerns for environment biodiversity.

Nabumetone and flufenamic acid pose a serious risk to aquatic plants: A study with Chlamydomonas reinhardtii as a model organism.

The aquatic environment is constantly under threat due to the release of numerous pollutants. Among them, pharmaceuticals constitute a huge and diverse group. Non-steroidal anti-inflammatory drugs (NSAIDs) are increasingly found in water bodies, but knowledge about their potential toxicity is still low. In particular, there is a lack of information about their influences on aquatic plants and algae. We estimated the susceptibility of the microalgae Chlamydomonas reinhardtii to nabumetone (NBT) and flufenamic acid (FFA), focusing on photosynthesis. Due to the differences in the structures of these compounds, it was assumed that these drugs would have different toxicities to the tested green algae. The hypothesis was confirmed by determining the effective concentration values, the intensity of photosynthesis, the intensity of dark respiration, the contents of photosynthetic pigments, the fluorescence of chlorophyll a in vivo (OJIP test), and cell ultrastructure analysis. Assessment of the toxicity of the NSAIDs was extended by the calculation of an integrated biomarker response index (IBR), which is a valuable tool in ecotoxicological studies. The obtained results indicate an over six times higher toxicity of NBT compared to FFA. After analysis of the chlorophyll a fluorescence in vivo, it was found that NBT inhibited electron transport beyond the PS II. FFA, unlike NBT, lowered the intensity of photosynthesis, probably transforming some reaction centers into "silent centers", which dissipate energy as heat. The IBR estimated based on photosynthetic parameters suggests that the toxic effect of FFA results mainly from photosynthesis disruption, whereas NBT significantly affects other cellular processes. No significant alteration in the ultrastructure of treated cells could be seen, except for changes in starch grain number and autophagic vacuoles that appeared in FFA-treated cells. To the best of our knowledge, this is the first work reporting the toxic effects of NBT and FFA on unicellular green algae.

Green alga Chlamydomonas reinhardtii can effectively remove diclofenac from the water environment - A new perspective on biotransformation.

The use of unicellular algae to remove xenobiotics (including drugs) from wastewaters is one of the rapidly developing areas of environmental protection. Numerous data indicate that for efficient phycoremediation three processes are important, i.e. biosorption, bioaccumulation, and biotransformation. Although biosorption and bioaccumulation do not raise any serious doubts, biotransformation is more problematic since its products can be potentially more toxic than the parent compounds posing a threat to organisms living in a given environment, including organisms that made this transformation. Thus, two questions need to be answered before the proper algae strain is chosen for phycoremediation, namely what metabolites are produced during biotransformation, and how resistant is the analyzed strain to a mixture of parent compound and metabolites that appear over the course of culture? In this work, we evaluated the remediation potential of the model green alga Chlamydomonas reinhardtii in relation to non-steroidal anti-inflammatory drugs (NSAIDs), as exemplified by diclofenac. To achieve this, we analysed the susceptibility of C. reinhardtii to diclofenac as well as its capability to biosorption, bioaccumulation, and biotransformation of the drug. We have found that even at a relatively high concentration of diclofenac the algae maintained their vitality and were able to remove (37.7%) DCF from the environment. A wide range of phase I and II metabolites of diclofenac (38 transformation products) was discovered, with many of them characteristic rather for animal and bacterial biochemical pathways than for plant metabolism. Due to such a large number of detected products, 18 of which were not previously reported, the proposed scheme of diclofenac transformation by C. reinhardtii not only significantly contributes to broadening the knowledge in this field, but also allows to suggest possible pathways of degradation of xenobiotics with a similar structure. It is worth pointing out that a decrease in the level of diclofenac in the media observed in this study cannot be fully explained by biotransformation (8.4%). The mass balance analysis indicates that other processes (total 22%), such as biosorption, a non-extractable residue formation, or complete decomposition in metabolic cycles can be involved in the diclofenac disappearance, and those findings open the prospects of further research.

Cross Talk between Hydrogen Peroxide and Nitric Oxide in the Unicellular Green Algae Cell Cycle: How Does It Work?

The regulatory role of some reactive oxygen species (ROS) and reactive nitrogen species (RNS), such as hydrogen peroxide or nitric oxide, has been demonstrated in some higher plants and algae. Their involvement in regulation of the organism, tissue and single cell development can also be seen in many animals. In green cells, the redox potential is an important photosynthesis regulatory factor that may lead to an increase or decrease in growth rate. ROS and RNS are important signals involved in the regulation of photoautotrophic growth that, in turn, allow the cell to attain the commitment competence. Both hydrogen peroxide and nitric oxide are directly involved in algal cell development as the signals that regulate expression of proteins required for completing the cell cycle, such as cyclins and cyclin-dependent kinases, or histone proteins and E2F complex proteins. Such regulation seems to relate to the direct interaction of these signaling molecules with the redox-sensitive transcription factors, but also with regulation of signaling pathways including MAPK, G-protein and calmodulin-dependent pathways. In this paper, we aim to elucidate the involvement of hydrogen peroxide and nitric oxide in algal cell cycle regulation, considering the role of these molecules in higher plants. We also evaluate the commercial applicability of this knowledge. The creation of a simple tool, such as a precisely established modification of hydrogen peroxide and/or nitric oxide at the cellular level, leading to changes in the ROS-RNS cross-talk network, can be used for the optimization of the efficiency of algal cell growth and may be especially important in the context of increasing the role of algal biomass in science and industry. It could be a part of an important scientific challenge that biotechnology is currently focused on.

Pharmaceuticals in the Aquatic Environment: A Review on Eco-Toxicology and the Remediation Potential of Algae.

The pollution of the aquatic environment has become a worldwide problem. The widespread use of pesticides, heavy metals and pharmaceuticals through anthropogenic activities has increased the emission of such contaminants into wastewater. Pharmaceuticals constitute a significant class of aquatic contaminants and can seriously threaten the health of non-target organisms. No strict legal regulations on the consumption and release of pharmaceuticals into water bodies have been implemented on a global scale. Different conventional wastewater treatments are not well-designed to remove emerging contaminants from wastewater with high efficiency. Therefore, particular attention has been paid to the phycoremediation technique, which seems to be a promising choice as a low-cost and environment-friendly wastewater treatment. This technique uses macro- or micro-algae for the removal or biotransformation of pollutants and is constantly being developed to cope with the issue of wastewater contamination. The aims of this review are: (i) to examine the occurrence of pharmaceuticals in water, and their toxicity on non-target organisms and to describe the inefficient conventional wastewater treatments; (ii) present cost-efficient algal-based techniques of contamination removal; (iii) to characterize types of algae cultivation systems; and (iv) to describe the challenges and advantages of phycoremediation.

Diclofenac Alters the Cell Cycle Progression of the Green Alga Chlamydomonas reinhardtii.

The aim of the study was to verify the hypothesis that a potential cause of the phytotoxicity of diclofenac (DCF, a non-steroidal anti-inflammatory drug) is an effect of cell cycle progression. This research was conducted using synchronous cultures of a model organism, green alga Chlamydomonas reinhardtii. The project examined DCF effects on selected parameters that characterize cell cycle progression, such as cell size, attainment of commitment points, DNA replication, number of nuclei formed during cells division and morphology of cells in consecutive stages of the cell cycle, together with the physiological and biochemical parameters of algae cells at different stages. We demonstrated that individual cell growth remained unaffected, whereas cell division was delayed in the DCF-treated groups grown in continuous light conditions, and the number of daughter cells from a single cell decreased. Thus, the cell cycle progression is a target affected by DCF, which has a similar anti-proliferative effect on mammalian cells.

Does diclofenac act like a photosynthetic herbicide on green algae? Chlamydomonas reinhardtii synchronous culture-based study with atrazine as reference.

The non-steroidal anti-inflammatory drug diclofenac (DCF) is one of the commonly used and frequently detected drugs in water bodies, and several studies indicate its toxic effect on plants and algae. Studies performed with asynchronous Chlamydomonas reinhardtii cultures indicated that DCF inhibit the growth of population of the algae. Here, a synchronous population of C. reinhardtii, in which all cells are in the same developmental phase, is used. Following changes in cells size, photosynthetic activity and gene expression, we could compare, at the level of single cell, DCF-mediated effects with the effects caused by atrazine, a triazine herbicide that inhibits photosynthesis and triggers oxidative stress. Application of DCF and atrazine at the beginning of the cell cycle allowed us to follow the changes occurring in the cells in the subsequent stages of their development. Synchronized Chlamydomonas reinhardtii cultures (strain CC-1690, wild type) were exposed to diclofenac sodium salt (135 mg/L) or atrazine (77.6 µg/L). The cell suspension was sampled hourly (0-10 h) in the light period of the cell cycle to determine cell number and volume, photosynthetic pigment content, chlorophyll a fluorescence (OJIP test) in vivo, and selected gene expression (real-time qPCR), namely psbA, psaA, FSD1, MSD3 and APX1. The two toxicants differently influenced C. reinhardtii cells. Both substances decreased photosynthetic "vitality" (PI - performance index) of the cells, albeit for different reasons. While atrazine significantly disrupted the photosynthetic electron transport, resulting in excessive production of reactive oxygen species (ROS) and limited cell growth, DCF caused silencing of photosystem II (PSII) reaction centers, transforming them into "heat sinks", thus preventing significant ROS overproduction. Oxidative stress caused by atrazine was the probable reason for the rapid appearance of phytotoxic action soon after entering the cells, while the effects of DCF could only be seen several hours after treatment. A comparison of DCF-caused effects with the effects caused by atrazine led us to conclude that, although DCF cannot be regarded as typical photosynthetic herbicide, it exhibits an algicidal activity and can be potentially dangerous for aquatic plants and algae.

Diclofenac and atrazine restrict the growth of a synchronous Chlamydomonas reinhardtii population via various mechanisms.

Non-steroidal anti-inflammatory drug diclofenac (DCF) is commonly found in freshwater bodies and can have adverse effects on non-target organisms. Among the studies on DCF toxicity, several ones have reported its harmful effects on plants and algae. To gain a better understanding of the mechanisms of DCF toxicity towards green algae, we used a synchronous Chlamydomonas reinhardtii cc-1690 culture and compared DCF (135 mg/L) effects with effects caused by atrazine (ATR; 77.6 µg/L), an herbicide with a well-known mechanism of toxic action. To achieve our goal, cell number and size, photosynthetic oxygen consumption/evolution, chlorophyll a fluorescence in vivo, H2O2 production by the cells, antioxidative enzymes encoding genes expression were analyzed during light phase of the cell cycle. We have found, that DCF and ATR affect C. reinhardtii through different mechanisms. ATR inhibited the photosynthetic electron transport chain and induced oxidative stress in chloroplast. Such chloroplastic energetics disruption indirectly influenced respiration, the intensification of which could partially mitigate low efficiency of photosynthetic energy production. As a result, ATR inhibited the growth of single cell leading to limitation in C. reinhardtii population development. In contrast to ATR-treated algae, in DCF-treated cells the fraction of active PSII reaction centers was diminished without drastic changes in electron transport or oxidative stress symptoms in chloroplast. However, significant increase in transcript level of gene encoding for mitochondria-located catalase indicates respiratory processes as a source of H2O2 overproduced in the DCF-treated cells. Because the single cell growth was not strongly affected by DCF, its adverse effect on progeny cell number seemed to be related rather to arresting of cell divisions. Concluding, although the DCF phytotoxic action appeared to be different from the action of the typical herbicide ATR, it can act as algal growth-inhibiting factor in the environment.

The effect of vanadium(IV) complexes on development of Arabidopsis thaliana subjected to H2O2-induced stress.

The impact of oxydiacetate oxidovanadium(IV) complexes on plants is currently unknown. This report demonstrates the influence of these complexes on Arabidopsis thaliana (L.) Heynh. In the presence of 10-6M vanadium(IV) complexes, plants proceeded through their entire life cycle, with the occurrence of proper morphological and cytological organisation of leaf and root tissues. The addition of 10-1M H2O2 caused root damage, leaf necrosis, and plant death at around the seventh day, due to the destruction of the root system. Pretreatment of the plants with 10-6M of vanadium(IV) compounds: VOSO4 and VO(oda), alleviated the effects of H2O2 to some extent. Plants pretreated with 10-6M vanadium(IV) complexes survived longer despite the presence of H2O2. Considering the higher rate of plant survival in the presence of VOSO4, and the relatively high photosynthetic parameters and anthocyanin contents in the cells, we conclude that this vanadium(IV) compound can have positive effects on plants that are grown under stress conditions.

Exogenously applied hydrogen peroxide modifies the course of the Chlamydomonas reinhardtii cell cycle.

The interaction of NO and H2O2 in the regulation of plant development is well documented. We have recently shown that the content of NO and H2O2 changes in a characteristic way during the cell cycle of Chlamydomonas reinhardtii (Pokora et al., 2017), which implies participation of these molecules in the regulation of Chlamydomonas development. To verify this assumption, H2O2 was supplied at a concentration about 1.5 times higher than that determined in the control cells. Cells were synchronized by alternating the light/dark (10/14 h) regimen. H2O2 was added to zoospore suspensions, previously held in the dark, and cells growing for 3, 6, and 9 h in the light. The data indicate that, depending on the phase of the Chlamydomonas cell cycle, H2O2, via mild modification of redox homeostasis, may: a) accelerate or delay the duration of the cell cycle; b) increase the number of replication rounds occurring in one cell cycle; c) modify the biomass and cell volume of progeny cells and d) accelerate the liberation of daughter cells. This provides a tool to control the development of Chlamydomonas cell and thus offers the opportunity to obtain a population of cells with characteristics desired in biotechnology.

Phytotoxic activity of diclofenac: Evaluation using a model green alga Chlamydomonas reinhardtii with atrazine as a reference substance.

Human activities have caused increasing inputs of pharmaceuticals to the environment and diclofenac (DF) is one of the most commonly detected in freshwater systems. The aim of this study was to determine the impact of DF on a freshwater green alga as a non-target organism. For DF toxicity evaluation, its effects on a model organism Chlamydomonas reinhardtii were compared with effects caused by the herbicide atrazine (AT). EC50 values were about 135 mg/L for DF and 78 mg/L for AT, respectively. Both toxicants enhanced H2O2 production by the cells (144% and 178% of control for AT and DF, respectively) and stimulated catalase activity (≈200% of control). Activity of ascorbate peroxidase was elevated in AT-cells but not in DF-treated cells. DF did not influence dark respiration of the cells, whereas AT inhibited this process by about 50% compared to the control. Both toxicants caused photosynthesis inhibition. Analysis of parameters of chlorophyll a fluorescence in vivo showed diminishment of a performance index (PI) in both DF- and AT-treated cells (≈50% of control), but the reasons for the changes detected were different. AT diminished the efficiency of electron transport between PS II and PS I without significant inhibition of PS II or PS I reaction centers (RCs). In contrast to AT, DF seemed to influence directly PS II RCs. The fraction of active PS II RCs was lowered in DF-treated cells, but energy flux per active RC increased. Our study indicates that DF phytotoxicity results mainly from photosynthesis inhibition due to "silencing" of a fraction of PS II RCs.

Changes in nitric oxide/hydrogen peroxide content and cell cycle progression: Study with synchronized cultures of green alga Chlamydomonas reinhardtii.

The present study aimed to evaluate the possible relationship between the changes in hydrogen peroxide (H2O2) and nitric oxide (NO) content and the course of growth and reproductive processes of the cell cycle of Chlamydomonas reinhardtii. The peak of H2O2 observed at the beginning of the cell cycle was found to originate from Fe-SOD and Mn-SODchl. activity and result from the alternation in the photosynthetic processes caused by the dark-to-light transition of daughter cells. A rapid increase in NO concentration, observed before the light-to-dark cell transition, originated from NR and NIR activity and was followed by a photosynthesis-independent, Mn-SODchl.-mediated increases in H2O2 production. This H2O2 peak overlapped the beginning of Chlamydomonas cell division, which was indicated by a profile of CYCs and CDKs characteristic of cells' passage through the G1/S and S/M checkpoints. Taken together, our results show that there is a clear relationship between the course of the Chlamydomonas cell cycle and typical changes in the H2O2/NO ratio, as well as changes in expression and activity of enzymes involved in generation and scavenging of these signaling molecules.

Time-dependent changes in antioxidative enzyme expression and photosynthetic activity of Chlamydomonas reinhardtii cells under acute exposure to cadmium and anthracene.

Heavy metals (HM) and polycyclic aromatic hydrocarbons (PAHs) are present in the freshwater environment at concentrations that can be hazardous to the biota. Among HMs and PAHs, cadmium (Cd) and anthracene (ANT) are the most prevalent and toxic ones. The response of Chlamydomonas cells to Cd and ANT at concentrations that markedly reduced the growth of algal population was investigated in this study. At such concentrations, both cadmium and anthracene were recognized as oxidative stress inducers, since high concentration of H2O2 in treated cultures was observed. Therefore, as a part of the "molecular phase" of the cell response to this stress, we examined the time-dependent expression of genes encoding the main antioxidative enzymes: superoxide dismutase (SOD), catalase (CAT) and ascorbate peroxidase (APX), as well as the activity of these enzymes in cells, with special attention paid to chloroplastic and mitochondrial isoforms of SOD. To characterize the cell response at the "physiological level", we examined the photosynthetic activity of stressed cells via analysis of chlorophyll a fluorescence in vivo. In contrast to standard ecotoxicity studies in which the growth end-points are usually determined, herein we present time-dependent changes in algal cell response to Cd- and ANT-induced stress. The most significant effect(s) of the toxicants on photosynthetic activity was observed in the 6th hour, when strong depression of PI parameter value, an over 50 percent reduction of the active reaction center fraction (RC0) and a 3-fold increase in non-photochemical energy dissipation (DI0/RC) were noted. At the same time, the increase (up to 2.5-fold) in mRNA transcript of SOD and CAT genes, followed by the enhancement in the enzyme activity was observed. The high expression of the Msd 3 gene in treated Chlamydomonas cells probably complements the partial loss of chloroplast Fe-SOD and APX activity, while catalase and Mn-SOD 5 seem to be the major enzymes responsible for mitochondrion protection. The progressive increase in SOD and CAT activities seems to be involved in the recovery of photosynthesis within 12-24h after the application of the toxicants.

The mechanism of anthracene interaction with photosynthetic apparatus: a study using intact cells, thylakoid membranes and PS II complexes isolated from Chlamydomonas reinhardtii.

Intact cells of Chlamydomonas reinhardtii as well as isolated thylakoid membranes and photosystem II complexes were used to examine a possible mechanism of anthracene (ANT) interaction with the photosynthetic apparatus. Since ANT concentrations above 1 mM were required to significantly inhibit the rate of oxygen evolution in PS II membrane fragments it may indicate that the toxicant did not directly interact with this photosystem. On the other hand, stimulation of oxygen uptake by ANT-treated thylakoids suggested that ANT could either act as an artificial electron acceptor in the photosynthetic electron transport chain or function as an uncoupler. Electron transfer from excited chlorophyll to ANT is impossible due to the very low reduction potential of ANT and therefore we propose that toxic concentrations of ANT increase the thylakoid membrane permeability and thereby function as an uncoupler, enhancing electron transport in vitro. Hence, its unspecific interference with photosynthetic membranes in vitro suggests that the inhibitory effect observed on intact cell photosynthesis is caused by uncoupling of phosphorylation.

Toxicity of cadmium, anthracene, and their mixture to Desmodesmus subspicatus estimated by algal growth-inhibition ISO standard test.

Cells of Desmodesmus subspicatus 86.81 were used to examine the toxicity of cadmium chloride (CdCl(2)) and anthracene (ANT) applied individually and in combination. The experiments were performed according to standardized ISO (International Organization for Standardization) 8692 protocol (2004). Parameters measured were the number of cells and chlorophyll a fluorescence parameters. E(r)C(10) and E(r)C(50) values (growth rate [r] inhibition by 10% and 50%, respectively) for single toxicants were determined separately. The effect of mixtures of the substances (Cd + ANT) at concentrations corresponding to E(r)C(10) (E(r)C(10) + E(r)C(10)) and E(r)C(50) (E(r)C(50) + E(r)C(50)) values was characterized. The toxicity of individual chemicals after a 72-h exposure was as follows: ANT (E(r)C(10) = 0.06; E(r)C(50) = 0.26 mg l(-1)) and CdCl(2) (E(r)C(10) = 0.12; E(r)C(50) = 0.30 mg l(-1)). The combination Cd + ANT decreased the population growth rate more strongly than the substances applied individually. Cadmium at a concentration corresponding to E(r)C(10) slightly influenced the parameters of chlorophyll a fluorescence as measured by the OJIP test (O, J, I, and P are the different steps of fluorescence induction curve), whereas the influence of ANT was not statistically significant. In Cd + ANT-treated samples, the photosynthetic "vitality" (PI), the maximum quantum yield of primary photochemistry (φ(Po)), and the fraction of active PS II reaction centre (RC) decreased, but the values of ABS/RC, TR(0)/RC, and DI(0)/RC increased. The type of interaction between Cd and ANT depended on the concentration of chemicals used. When the substances were applied at concentrations of E(r)C(10), synergistic effects were observed, whereas the mixture of chemicals used at an E(r)C(50) concentration showed an antagonistic interaction.

A metabolomic approach to study major metabolite changes during acclimation to limiting CO2 in Chlamydomonas reinhardtii.

Using a gas chromatography-mass spectrometry-time of flight technique, we determined major metabolite changes during induction of the carbon-concentrating mechanism in the unicellular green alga Chlamydomonas reinhardtii. In total, 128 metabolites with significant differences between high- and low-CO(2)-grown cells were detected, of which 82 were wholly or partially identified, including amino acids, lipids, and carbohydrates. In a 24-h time course experiment, we show that the amino acids serine and phenylalanine increase transiently while aspartate and glutamate decrease after transfer to low CO(2). The biggest differences were typically observed 3 h after transfer to low-CO(2) conditions. Therefore, we made a careful metabolomic examination at the 3-h time point, comparing low-CO(2) treatment to high-CO(2) control. Five metabolites involved in photorespiration, 11 amino acids, and one lipid were increased, while six amino acids and, interestingly, 21 lipids were significantly lower. Our conclusion is that the metabolic pattern during early induction of the carbon-concentrating mechanism fit a model where photorespiration is increasing.

Intact anthracene inhibits photosynthesis in algal cells: a fluorescence induction study on Chlamydomonas reinhardtii cw92 strain.

Short-term (24h) experiments were performed to examine the effect of anthracene (ANT) on Chlamydomonas reinhardtii cw92 grown in a batch culture system aerated with 2.5% CO(2). At concentrations ranging from 0.7 to 5.6 microM, ANT inhibited the growth of population in a concentration-dependent manner and EC(50) calculated amounted to 1.6 microM. At concentrations from 0.7 to 4.2 microM ANT stimulated respiration and inhibited the intensity of photosynthesis but did not affect chlorophyll content in the cells. ANT influenced chlorophyll a fluorescence parameters, measured by OJIP test (O, J, I and P are the different steps of fluorescence induction curve). ANT diminished the performance index (PI), the yield of primary photochemistry (phi(Po)), the yield of electron transport (phi(Epsilonomicron), the efficiency of moving the electron beyond Qa(-) (Psi(0)) and the fraction of active oxygen evolving complexes (OEC). The fraction of active PS II reaction centres in the treated samples dramatically dropped. The most pronounced changes in ANT-treated cells were observed in the stimulation of energy dissipation parameter (DI(0)/RC). The only OJIP parameter that was not influenced by ANT was energy absorption by photosynthetic antennae (ABS). The results lead to a conclusion that the inhibition of photosynthesis may be a consequence of unspecific ANT-membrane interaction, resulting from hydrophobic character of this hydrocarbon.