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.

Evaluation of Analytes Characterized with Potential Protective Action after Rat Exposure to Lead.

Lead (Pb) was revealed for its role as a neurodevelopmental toxin. The determination of neurotransmitters (NTs) in particular brain regions could ameliorate the precise description and optimization of therapeutic protocols able to restore the harmony of signaling pathways in nervous and immune systems. The determination of selected analytes from the group of NTs based on the liquid chromatography (LC)-based method was carried out to illustrate the changes of amino acid (AA) and biogenic amine (BA) profiles observed in chosen immune and nervous systems rat tissues after Pb intoxication. Also, a protective combination of AA was proposed to correct the changes caused by Pb intoxication. After the administration of Pb, changes were observed in all organs studied and were characterized by a fluctuation of NT concentrations in immune and nervous systems (hypothalamus samples). Using a protective mixture of bioactive compounds prevented numerous changes in the balance of NT. The combined analysis of the immune and nervous system while the normalizing effect of curative agents on the level of differentially secreted NTs and AA is studied could present a new approach to the harmonization of those two essential systems after Pb intoxication.

Comparison of Three Extraction Approaches for the Isolation of Neurotransmitters from Rat Brain Samples.

The determination of neurotransmitters (NTs) as relevant potential biomarkers in the study of various central nervous system (CNS) pathologies has been demonstrated. Knowing that NTs-related diseases mostly occupy individual regions of the nervous system, as observed, for instance, in neurodegenerative diseases (Alzheimer's and Parkinson's Diseases), the analysis of brain slices is preferred to whole-brain analysis. In this report, we present sample preparation approaches, such as solid-phase extraction, solid-phase microextraction, and dispersive liquid⁻liquid microextraction, and discuss the pitfalls and advantages of each extraction method. The ionic liquid (1-ethyl-3-methylimidazolium tetrafluoroborate)-assisted solid-phase microextraction (IL-SPME) is found to be, in our research, the relevant step towards the simultaneous determination of six NTs, namely, dopamine (DA), adrenaline (A), noradrenaline (NA), serotonin (5-HT), l-tryptophan (l-Trp), l-tyrosine (l-Tyr) in rat brain samples. The development of a novel bioanalytical technique for the evaluation of biomarkers in the context of green chemistry might be accelerated just with the use of IL, and this approach can be considered an advantageous strategy.

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.

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.

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.

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.

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.