Impact of anthracycline-based chemotherapy on RB1 gene methylation in peripheral blood leukocytes and biomarkers of oxidative stress and inflammation in sarcoma patients.

PURPOSE: We investigated the impact of anthracycline-based chemotherapy on methylation status of RB1 gene in peripheral blood leukocytes together with parameters of oxidative stress and inflammation in sarcoma patients. PATIENTS/METHODS: Blood samples were collected from 51 consecutive newly diagnosed sarcoma patients admitted to University Hospital Center Zagreb (Zagreb, Croatia) for first-line chemotherapy before the first cycle and post-chemotherapy. Methylation and copy number variation (CNV) of leukocyte RB1 gene were assessed using MS-MLPA probes. In addition, in blood samples, parameters of oxidative stress (ROS, MDA, SOD, and GSH) and inflammation (CRP, WBC, and NBC) were followed. RESULTS: In pre-chemotherapy samples, no CNVs and aberrant methylation of CpG106 promoter region of RB1 gene were detected; however, one patient had hypermethylation (by approximately 10%) of imprinted locus CpG85 in intron 2 of RB1 gene. In addition, a very good correlation of the tumor burden and CRP and tumor burden and GSH was found. The anthracycline-based chemotherapy reverts methylation of RB1 gene-imprinted locus CpG85 to normal level. Moreover, inflammation and oxidative stress parameters such as CRP, WBC, ROS, and MDA were significantly decreased in post-chemotherapy samples. CONCLUSION: This single-centered study on a cohort of consecutive sarcoma patients indicates that sarcoma patients can have aberrant germline DNA methylation and confirms the relationship of tumor burden with inflammation and oxidative stress. The applied chemotherapy protocols reverted RB1 gene methylation to normal level and decreased the level of inflammation and oxidative damage, thus indicating chemotherapy benefit to the patient's health status.

Phytotoxic Action of Silver Nanoparticles on Lemna minor: Multi-Parameter Analysis of Different Physiological Processes.

Considering the widespread use of silver nanoparticles (AgNPs) and their consequent build-up in waterways, there is a concern about the hazardous effect of AgNPs for aquatic ecosystems. The aim of this study was to clarify the mechanism of the action of AgNPs on duckweed (Lemna minor L.) by evaluating multiple parameters in different physiological processes. Duckweed was treated with AgNPs in a concentration range of 0.5 to 5 mg/L over a 7-day period. The analysis revealed that the AgNP-treated duckweed accumulated Ag in accordance with increasing AgNP concentrations. Furthermore, higher concentrations (2 and 5 mg/L) of AgNPs negatively affected N, P and especially K and Mg levels in the plant tissue. Accordingly, the plant growth and photosynthetic parameters were more inhibited in response to higher concentrations of AgNPs. Nanosilver significantly increased the generation of ROS at higher concentrations, although lipid peroxidation was significant even at the lowest concentration of AgNPs. However, defense mechanisms were able to counteract AgNP-induced oxidative stress and balance the intracellular redox status, as evidenced by increased activities of the main detoxification enzymes. With this experimental setting, AgNPs exhibited a relatively weak phytotoxicity at 0.5 and 1 mg/L; nevertheless, silver in a nano form poses a hazard for plants, considering its continuous release into aquatic environments.

Combining short-term bioassays using fish and crustacean model organisms with ToxCast in vitro data and broad-spectrum chemical analysis for environmental risk assessment of the river water (Sava, Croatia).

This study focused on the short-term whole organism bioassays (WOBs) on fish (Danio rerio) and crustaceans (Gammarus fossarum and Daphnia magna) to assess the negative biological effects of water from the major European River Sava and the comparison of the obtained results with in vitro toxicity data (ToxCast database) and Risk Quotient (RQ) methodology. Pollution profiles of five sampling sites along the River Sava were assessed by simultaneous chemical analysis of 562 organic contaminants (OCs) of which 476 were detected. At each sampling site, pharmaceuticals/illicit drugs category was mostly represented by their cumulative concentration, followed by categories industrial chemicals, pesticides and hormones. An exposure-activity ratio (EAR) approach based on ToxCast data highlighted steroidal anti-inflammatory drugs, antibiotics, antiepileptics/neuroleptics, industrial chemicals and hormones as compounds with the highest biological potential. Summed EAR-based prediction of toxicity showed a good correlation with the estimated toxicity of assessed sampling sites using WOBs. WOBs did not exhibit increased mortality but caused various sub-lethal biological responses that were dependant relative to the sampling site pollution intensity as well as species sensitivity. Exposure of G. fossarum and D. magna to river water-induced lower feeding rates increased GST activity and TBARS levels. Zebrafish D. rerio embryo exhibited a significant decrease in heartbeat rate, failure in pigmentation formation, as well as inhibition of ABC transporters. Nuclear receptor activation was indicated as the biological target of greatest concern based on the EAR approach. A combined approach of short-term WOBs, with a special emphasis on sub-lethal endpoints, and chemical characterization of water samples compared against in vitro toxicity data from the ToxCast database and RQs can provide a comprehensive insight into the negative effect of pollutants on aquatic organisms.

Grapevine Leafroll-Associated Virus 3 in Single and Mixed Infections Triggers Changes in the Oxidative Balance of Four Grapevine Varieties.

With the aim to characterize changes caused by grapevine leafroll-associated virus 3 (GLRaV-3) singly or in coinfection with other viruses and to potentially determine genotype-specific or common markers of viral infection, thirty-six parameters, including nutrient status, oxidative stress parameters, and primary metabolism as well as symptoms incidence were investigated in 'Cabernet Franc,' 'Merlot,' 'Pinot Noir,' and 'Tribidrag' grapevine varieties. Host responses were characterized by changes in cellular redox state rather than disturbances in nutrient status and primary metabolic processes. Superoxide dismutase, hydrogen peroxide, and proteins were drastically affected regardless of the type of isolate, the host, and the duration of the infection, so they present cellular markers of viral infection. No clear biological pattern could be ascertained for each of the GLRaV-3 genotypes. There is a need to provide a greater understanding of virus epidemiology in viticulture due to the increasing natural disasters and climate change to provide for global food production security. Finding grape varieties that will be able to cope with those changes can aid in this task. Among the studied grapevine varieties, autochthonous 'Tribidrag' seems to be more tolerant to symptoms development despite numerous physiological changes caused by viruses.

Physiological and Biochemical Response of Wild Olive (Olea europaea Subsp. europaea var. sylvestris) to Salinity.

In the face of climate change, water deficit and increasing soil salinity pose an even greater challenge to olive cultivation in the Mediterranean basin. Due to its tolerance to abiotic stresses, wild olive (Olea europaea subsp. europaea var. sylvestris) presents a good candidate in breeding climate-resilient olive varieties. In this study, the early response of the native Croatian wild olive genotype (WOG) to salinity was evaluated and compared with that of well-known cultivars (cv.) Leccino and Koroneiki. Potted olive plants were exposed either to 150 mM NaCl or 300 mM mannitol for 3 weeks to distinguish between the osmotic and ionic components of salt stress. To determine the impact of the plant age on salinity, 1-, 2-, and 3-year-old WOG plants were used in the study. The growth parameters of both the cultivars and WOG of different ages decreased in response to the mannitol treatment. In contrast to cv. Leccino, the NaCl treatment did not significantly affect the growth of cv. Koroneiki or WOG of any age. The contents of Na+ and Cl- were considerably higher in the salt-treated WOG, regardless of age, compared with the cultivars. However, while both treatments significantly reduced the K+ content of cv. Koroneiki, that nutrient was not significantly affected in either cv. Leccino or WOG. Unlike the cultivars and older WOG, the NaCl treatment caused a significant decline of photosynthetic pigments in the 1-year-old WOG. The cultivars and WOG of different ages experienced a similar drop in the chlorophyll a content under the isotonic mannitol treatment. The absence of lipid peroxidation, modulation of superoxide dismutase, and guaiacol peroxidase activity were noted in all WOG ages under both stressors. These data suggest that WOG resilience to salinity is associated with its large leaf capacity for Na+ and Cl- accumulation, K+ retention, and its adaptable antioxidative mechanisms. The results are promising with regard to obtaining a new olive cultivar with better resilience to soil salinity.

Gold and silver nanoparticles effects to the earthworm Eisenia fetida - the importance of tissue over soil concentrations.

To address and to compare the respective impact of gold and silver nanoparticles (Au and Ag NPs) in soil invertebrate, the earthworm Eisenia fetida was exposed to soil containing 2, 10, and 50 mg/kg of Au and Ag in both nanoparticulate and ionic forms for 10 days. Both metal NPs were 2-15 times less bioavailable than their ionic forms, and displayed similar transfer coefficients from soil to earthworm tissues. Both metal NPs triggered the onset of an oxidative stress as illustrated by increased glutathione S-transferase levels, decreased catalase levels, and increased malondialdehyde concentrations. Protein carbonylation distinguished the nanoparticular from the ionic forms as its increase was observed only after exposure to the highest concentration of both metal NPs. Au and Ag NPs triggered DNA modifications even at the lowest concentration, and both repressed the expression of genes involved in the general defense and stress response at high concentrations as did their ionic counterparts. Despite the fact that both metal NPs were less bioavailable than their ionic forms, at equivalent concentrations accumulated within earthworms tissues they exerted equal or higher toxic potential than their ionic counterparts.Capsule: At equivalent concentrations accumulated within earthworm tissues Au and Ag NPs exert equal or higher toxic potential than their ionic forms.

Correlations between Phytohormones and Drought Tolerance in Selected Brassica Crops: Chinese Cabbage, White Cabbage and Kale.

Drought is one of the major abiotic stresses affecting the productivity of Brassica crops. To understand the role of phytohormones in drought tolerance, we subjected Chinese cabbage (B. rapa ssp. pekinensis), white cabbage (B. oleracea var. capitata), and kale (B. oleracea var. acephala) to drought and examined the stress response on the physiological, biochemical and hormonal levels. The phytohormones abscisic acid (ABA), auxin indole-3-acetic acid (IAA), brassinosteroids (BRs), cytokinins (CKs), jasmonates (JAs), and salicylic acid (SA) were analyzed by ultra-high-performance liquid chromatography⁻tandem mass spectrometry (UHPLC-MS/MS). Based on the physiological and biochemical markers the Chinese cabbage exhibited the lowest tolerance, followed by the white cabbage, while the kale appeared to be the most tolerant to drought. The drought tolerance of the kale correlated with increased levels of SA, ABA, IAA, CKs iP(R) and cZ(R), and typhasterol (TY), a precursor of active BRs. In contrast, the drought sensitivity of the Chinese cabbage correlated with a significant increase in ABA, JAs and the active BRs castasterol (CS) and brassinolide (BL). The moderately tolerant white cabbage, positioned between the kale and Chinese cabbage, showed more similarity in terms of the phytohormone patterns with the kale. We concluded that the drought tolerance in Brassicaceae is mostly determined by the increased endogenous levels of IAA, CKs, ABA and SA and the decreased levels of active BRs.

Biological Activity of Flavonoids and Rare Sesquiterpene Lactones Isolated From Centaurea ragusina L.

The endemic Croatian species Centaurea ragusina L., like other species from the genus Centaurea, has been traditionally used in Croatia as an antibacterial agent and for the treatment of gastrointestinal and urogenital disorders. In several chromatographic steps, three flavonoids and three sesquiterpene lactones (STLs) were isolated and identified from the most active fractions of the ethanol extract. Two STLs, one for which we created the trivial name ragusinin, and hemistepsin A are here reported for the first time as constituents of the genus Centaurea. All six compounds were screened for their effect on several tumor and one normal cell lines. Among them, ragusinin showed the best bioactivity and high specificity to affect tumor murine SCCVII, human HeLa and Caco-2 cell lines, but not the viability of normal V79 fibroblasts. Due to these characteristics the action of ragusinin was investigated in more detail. Since DNA is the primary target for many drugs with antibacterial and anticancer activity, we studied its interaction with ragusinin. Rather moderate binding affinity to DNA excluded it as the primary target of ragusinin. Due to the possibility of STL interaction with glutathione (GSH), the ubiquitous peptide that traps reactive compounds and other xenobiotics to prevent damage to vital proteins and nucleic acids, its role in deactivation of ragusinin was evaluated. Addition of the GSH precursor N-acetyl-cysteine potentiated the viability of HeLa cells, while the addition of GSH inhibitor L-buthionine sulfoximine decreased it. Moreover, pre-treatment of HeLa cells with the inhibitor of glutathione-S-transferase decreased their viability indicating the detoxifying role of GSH in ragusinin treated cells. Cell death, derived by an accumulation of cells in a G2 phase of the cell cylce, was shown to be independent of poly (ADP-ribose) polymerase and caspase-3 cleavage pointing toward an alternative cell death pathway.

Short-term salt stress in Brassica rapa seedlings causes alterations in auxin metabolism.

Salinity is one of major abiotic stresses affecting Brassica crop production. Here we present investigations into the physiological, biochemical, and hormonal components of the short-term salinity stress response in Chinese cabbage seedlings, with particular emphasis on the biosynthesis and metabolism of auxin indole-3-acetic acid (IAA). Upon salinity treatments (50-200 mM NaCl) IAA level was elevated in a dose dependent manner reaching 1.6-fold increase at the most severe salt treatment in comparison to the control. IAA precursor profiling suggested that salinity activated the indole-3-acetamide and indole-3-acetaldoxime biosynthetic pathways while suppressing the indole-3-pyruvic acid pathway. Levels of the IAA catabolites 2-oxoindole-3-acetic acid and indole-3-acetic acid-aspartate increased 1.7- and 2.0-fold, respectively, under the most severe treatment, in parallel with those of IAA. Conversely, levels of the ester conjugate indole-3-acetyl-1-O-ß-d-glucose and its catabolite 2-oxoindole-3-acetyl-1-O-ß-d-glucose decreased 2.5- and 7.0-fold, respectively. The concentrations of stress hormones including jasmonic acid and jasmonoyl-isoleucine (JA and JA-Ile), salicylic acid (SA) and abscisic acid (ABA) confirmed the stress induced by salt treatment: levels of JA and JA-Ile increased strongly under the mildest treatment, ABA only increased under the most severe treatment, and SA levels decreased dose-dependently. These hormonal changes were related to the observed changes in biochemical stress markers upon salt treatments: reductions in seedling fresh weight and root growth, decreased photosynthesis rate, increased levels of reactive oxygen species, and elevated proline content and the Na+/K+ ratio. Correlations among auxin profile and biochemical stress markers were discussed based on Pearson's coefficients and principal component analysis (PCA).

Phytochemical and Bioactive Potential of in vivo and in vitro Grown Plants of Centaurea ragusina L. - Detection of DNA/RNA Active Compounds in Plant Extracts via Thermal Denaturation and Circular Dichroism.

INTRODUCTION: The phytochemical composition and biological activity of non-volatile components of Centaurea ragusina L. has not been studied previously. OBJECTIVES: Our aim was to evaluate the phytochemical and bioactive potential (including interactions with polynucleotides) of C. ragusina L. depending on the origin of plant material (in vivo - leaves from natural habitats, ex vitro - leaves from plants acclimated from culture media, in vitro - leaves and calli from plants grown in culture media) and polarity of solvents used in extract preparation (80 and 96% ethanol and water combinations or single solvents). METHODOLOGY: The polyphenol composition was determined by spectrophotometric and HPLC analysis. Biological activity of extracts was evaluated by following methods: 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS) and 1,1-diphenyl-2-picrylhydrazyl (DPPH) methods for antioxidative activity, 2,3,5-triphenyl tetrazolium chloride (TTC) microdilution method for antibacterial activity, crystal-violet test for cytotoxic activity and thermal denaturation (TD) and circular dichroism (CD) for DNA/RNA interactions. RESULTS: Conditions for the most efficient polyphenol extraction were determined: the 80% ethanol/water solvent system was the most suitable for callus and leaf ex vitro samples and 80 or 96% ethanol for leaf in vivo samples. Significantly higher levels of chlorogenic acid and naringenin were detected in callus tissue than in vivo plant. Ethanolic extracts exhibited the significant antibacterial activity against Staphylococcus aureus ATCC 25923. DNA/RNA active compounds in plant extracts were detected by TD and CD methods. CONCLUSIONS: Callus tissue and ex vitro leaves represent a valuable source of polyphenols as in vivo leaves. TD and CD can be applied for detection of DNA/RNA active compounds in extracts from natural resources. Copyright © 2017 John Wiley & Sons, Ltd.

Quantification of malondialdehyde by HPLC-FL - application to various biological samples.

Malondialdehyde (MDA) is stabile product of lipid peroxidation (LPO), and therefore MDA is frequently used as a biomarker of LPO. To determine MDA level in various biological samples (human plasma, fish liver tissue and cells in culture), we used an HPLC method with fluorescent detection based on 2-thiobarbituric acid (TBA) assay. The method was validated by the use of spiked pooled plasma samples. In tested concentration range (0.15-3.0 µmol/L) the method was linear (R(2) = 0.9963), the between-day variability (coefficient of variations, CVs) was between 4.7 and 7.6%, the within-day variability CVs was between 2.6 and 6.4% and recovery was between 91.2 and 107.6%. The level of MDA in human plasma (healthy male, non-smokers, 46.3 ± 4.7 years; N = 38) was 2.2 ± 1.4 µmol/L; that in liver tissue of common carp (Cyprinus carpio; N = 12) was 0.02 ± 0.004 µmol/g tissue, and in cultured cells (human laryngeal carcinoma cells; N = 10) it was 0.18 ± 0.02 nmol/mg proteins. The HPLC-FL method is rapid, accurate and reliable to follow the extent of LPO in various biological samples, particularly in samples in which a low level of MDA is expected, such as cells in culture. Owing to the rapid analytical process and run time, it can be used for routine analysis of MDA in clinical laboratory.

Toxicological characterization of the landfill leachate prior/after chemical and electrochemical treatment: a study on human and plant cells.

In this research, toxicological safety of two newly developed methods for the treatment of landfill leachate from the Piskornica (Croatia) sanitary landfill was investigated. Chemical treatment procedure combined chemical precipitation with CaO followed by coagulation with ferric chloride and final adsorption by clinoptilolite. Electrochemical treatment approach included pretreatment with ozone followed by electrooxidation/electrocoagulation and final polishing by microwave irradiation. Cell viability of untreated/treated landfill leachate was examined using fluorescence microscopy. Cytotoxic effect of the original leachate was obtained for both exposure periods (4 and 24 h) while treated samples showed no cytotoxic effect even after prolonged exposure time. The potential DNA damage of the untreated/treated landfill leachate was evaluated by the comet assay and cytokinesis-block micronucleus (CBMN) assay using either human or plant cells. The original leachate exhibited significantly higher comet assay parameters compared to negative control after 24 h exposure. On the contrary, there was no significant difference between negative control and chemically/electrochemically treated leachate for any of the parameters tested. There was also no significant increase in either CBMN assay parameter compared to the negative control following the exposure of the lymphocytes to the chemically or electrochemically treated landfill leachate for both exposure periods while the original sample showed significantly higher number of micronuclei, nucleoplasmic bridges and nuclear buds for both exposure times. Results suggest that both methods are suitable for the treatment of such complex waste effluent due to high removal efficiency of all measured parameters and toxicological safety of the treated effluent.