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.

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.

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.

Adaptation strategies of two closely related Desmodesmus armatus (green alga) strains contained different amounts of cadmium: a study with light-induced synchronized cultures of algae.

During the Desmodesmus armatus cell cycle, 8-celled coenobia of 276-4d strain accumulated a much lower amounts of cadmium than unicells of B1-76 strain. Cadmium reduced growth and photosynthesis in the cells of strain B1-76, but not those of 276-4d strain. Cells of 276-4d strain revealed a higher activity of superoxide dismutase (SOD) isoforms, in particular the activity and protein content of Fe-SOD. Cu/Zn-SOD was earlier and much stronger induced by cadmium in 276-4d than in B1-76 strain, whereas Fe- and Mn-SOD activity and Fe-SOD synthesis were induced only in 276-4d strain. Cadmium did not affect the heat shock protein 70 synthesis in B1-76 strain, but significantly stimulated this process in 276-4d strain. The level of glutathione increased 30-fold during cell development of Cd-exposed 276-4d strain, while in B1-76 it increased about 12 timed. Matured cells of both strains exposed to cadmium produced comparable amounts of phytochelatins and other thiol peptides, but their production in young cells of B1-76 strain was much higher than in 276-4d strain. In conclusion, a complex of internal detoxification mechanisms appeared to be more efficient in cells of 276-4d strain than B1-76 one.

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.