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.

Serum free fatty acid levels and insulin resistance in patients undergoing one-anastomosis gastric bypass.

INTRODUCTION: One anastomosis gastric bypass (OAGB) leads to improvement in glucose homeostasis; however, the mechanism of this beneficial effect is not fully understood. Increased serum free fatty acid (FFA) concentrations in obese subjects contribute to the development of insulin resistance and type 2 diabetes. AIM: The authors hypothesized that improvement in glucose homeostasis after OAGB may be associated with a decrease in FFA concentration. MATERIAL AND METHODS: Serum FFA levels were measured by gas chromatography-mass spectrometry before and 3 months after OAGB and, for comparison, in patients who underwent laparoscopic sleeve gastrectomy (LSG). Serum insulin was assayed by immunoenzymatic method, and other parameters by standard laboratory methods. RESULTS: OAGB resulted in a large decrease in FFA levels and great improvement in insulin sensitivity. These effects in patients after LSG were less prominent. CONCLUSIONS: Results suggest that decreased serum FFA levels after OAGB contribute to resolution of insulin sensitivity after this type of bariatric surgery.

One-anastomosis gastric bypass modulates the serum levels of pro- and anti-inflammatory oxylipins, which may contribute to the resolution of inflammation.

BACKGROUND/OBJECTIVES: Oxylipins are polyunsaturated fatty acid derivatives involved in the regulation of various processes, including chronic inflammation, insulin resistance and hepatic steatosis. They can be synthesized in various tissues, including adipose tissue. There is some evidence that obesity is associated with the deregulation of serum oxylipin levels. The aim of this study was to evaluate the effect of bariatric surgery (one-anastomosis gastric bypass) on the serum levels of selected oxylipins and their fatty acid precursors and to verify the hypothesis that their changes after surgery can contribute to the resolution of inflammation. Moreover, we compared the oxylipin levels (prostaglandin E2, 13-HODE, maresin 1 and resolvin E1), fatty acids and the expression of enzymes that synthesize oxylipins in adipose tissue of lean controls and subjects with severe obesity. SUBJECTS/METHODS: The study included 50 patients with severe obesity that underwent bariatric surgery and 41 subjects in lean, control group. Fatty acid content was analyzed by GC-MS, oxylipin concentrations were measured with immunoenzymatic assay kits and real-time PCR analysis was used to assess mRNA levels in adipose tissue. RESULTS: Our results show increased expression of some enzymes that synthesize oxylipins in adipose tissue and alterations in the levels of oxylipins in both adipose tissue and serum of subjects with obesity. After bariatric surgery, the levels of anti-inflammatory oxylipins increased, whereas pro-inflammatory oxylipins decreased. CONCLUSIONS: In patients with obesity, the metabolism of oxylipins is deregulated in adipose tissue, and their concentrations in serum are altered. Bariatric surgery modulates the serum levels of pro- and anti-inflammatory oxylipins, which may contribute to the resolution of inflammation.

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.

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.

Sample Preparation Methods for Lipidomics Approaches Used in Studies of Obesity.

Obesity is associated with alterations in the composition and amounts of lipids. Lipids have over 1.7 million representatives. Most lipid groups differ in composition, properties and chemical structure. These small molecules control various metabolic pathways, determine the metabolism of other compounds and are substrates for the syntheses of different derivatives. Recently, lipidomics has become an important branch of medical/clinical sciences similar to proteomics and genomics. Due to the much higher lipid accumulation in obese patients and many alterations in the compositions of various groups of lipids, the methods used for sample preparations for lipidomic studies of samples from obese subjects sometimes have to be modified. Appropriate sample preparation methods allow for the identification of a wide range of analytes by advanced analytical methods, including mass spectrometry. This is especially the case in studies with obese subjects, as the amounts of some lipids are much higher, others are present in trace amounts, and obese subjects have some specific alterations of the lipid profile. As a result, it is best to use a method previously tested on samples from obese subjects. However, most of these methods can be also used in healthy, nonobese subjects or patients with other dyslipidemias. This review is an overview of sample preparation methods for analysis as one of the major critical steps in the overall analytical procedure.

Decreased Triacylglycerol Content and Elevated Contents of Cell Membrane Lipids in Colorectal Cancer Tissue: A Lipidomic Study.

Recent evidence suggests that lipid composition in cancer tissues may undergo multiple alterations. However, no comprehensive analysis of various lipid groups in colorectal cancer (CRC) tissue has been conducted thus far. To address the problem in question, we determined the contents of triacylglycerols (TG), an energetic substrate, various lipids necessary for cell membrane formation, among them phospholipids (phosphatidylcholine, phosphatidylethanolamine), sphingolipids (sphingomyelin) and cholesterol (free, esterified and total), and fatty acids included in complex lipids. 1H-nuclear magnetic resonance (1H-NMR) and gas chromatography-mass spectrometry (GC-MS) were used to analyze the lipid composition of colon cancer tissue and normal large intestinal mucosa from 25 patients. Compared with normal tissue, cancer tissues had significantly lower TG content, along with elevated levels of phospholipids, sphingomyelin, and cholesterol. Moreover, the content of oleic acid, the main component of TG, was decreased in cancer tissues, whereas the levels of saturated fatty acids and polyunsaturated fatty acids (PUFAs), which are principal components of polar lipids, were elevated. These lipidome rearrangements were associated with the overexpression of genes associated with fatty acid oxidation, and the synthesis of phospholipids and cholesterol. These findings suggest that reprogramming of lipid metabolism might occur in CRC tissue, with a shift towards increased utilization of TG for energy production and enhanced synthesis of membrane lipids, necessary for the rapid proliferation of cancer cells.

The Effect of a High-Fat Diet on the Fatty Acid Composition in the Hearts of Mice.

The Western diet can lead to alterations in cardiac function and increase cardiovascular risk, which can be reproduced in animal models by implementing a high-fat diet (HFD). However, the mechanism of these alterations is not fully understood and may be dependent on alterations in heart lipid composition. The aim of this study was to evaluate the effect of an HFD on the fatty acid (FA) composition of total lipids, as well as of various lipid fractions in the heart, and on heart function. C57BL/6 mice were fed an HFD or standard laboratory diet. The FA composition of chow, serum, heart and skeletal muscle tissues was measured by gas chromatography-mass spectrometry. Cardiac function was evaluated by ultrasonography. Our results showed an unexpected increase in polyunsaturated FAs (PUFAs) and a significant decrease in monounsaturated FAs (MUFAs) in the heart tissue of mice fed the HFD. For comparison, no such effects were observed in skeletal muscle or serum samples. Furthermore, we found that the largest increase in PUFAs was in the sphingolipid fraction, whereas the largest decrease in MUFAs was in the phospholipid and sphingomyelin fractions. The hearts of mice fed an HFD had an increased content of triacylglycerols. Moreover, the HFD treatment altered aortic flow pattern. We did not find significant changes in heart mass or oxidative stress markers between mice fed the HFD and standard diet. The above results suggest that alterations in FA composition in the heart may contribute to deterioration of heart function. A possible mechanism of this phenomenon is the alteration of sphingolipids and phospholipids in the fatty acid profile, which may change the physical properties of these lipids. Since phospho- and sphingolipids are the major components of cell membranes, alterations in their structures in heart cells can result in changes in cell membrane properties.

Methods of the Analysis of Oxylipins in Biological Samples.

Oxylipins are derivatives of polyunsaturated fatty acids and due to their important and diverse functions in the body, they have become a popular subject of studies. The main challenge for researchers is their low stability and often very low concentration in samples. Therefore, in recent years there have been developments in the extraction and analysis methods of oxylipins. New approaches in extraction methods were described in our previous review. In turn, the old analysis methods have been replaced by new approaches based on mass spectrometry (MS) coupled with liquid chromatography (LC) and gas chromatography (GC), and the best of these methods allow hundreds of oxylipins to be quantitatively identified. This review presents comparative and comprehensive information on the progress of various methods used by various authors to achieve the best results in the analysis of oxylipins in biological samples.

Modern Methods of Sample Preparation for the Analysis of Oxylipins in Biological Samples.

Oxylipins are potent lipid mediators derived from polyunsaturated fatty acids, which play important roles in various biological processes. Being important regulators and/or markers of a wide range of normal and pathological processes, oxylipins are becoming a popular subject of research; however, the low stability and often very low concentration of oxylipins in samples are a significant challenge for authors and continuous improvement is required in both the extraction and analysis techniques. In recent years, the study of oxylipins has been directly related to the development of new technological platforms based on mass spectrometry (LC-MS/MS and gas chromatography-mass spectrometry (GC-MS)/MS), as well as the improvement in methods for the extraction of oxylipins from biological samples. In this review, we systematize and compare information on sample preparation procedures, including solid-phase extraction, liquid-liquid extraction from different biological tissues.

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.