Toxicity of TiO2 nanoparticles modified with dihydroquercetin

Cilj ovog rada je modifikacija površine nanočestica TiO2 bioaktivnim ligandima, kao što je dihidrokvercetin (DHQ), formiranjem interfacijalnog kompleksa sa prenosom naelektrisanja, kako bi se postigla njegova aktivacija pod vidljivom svetlošću. Neorganskoorganski hibridni nanokompozit TiO2 /DHQ okarakterisan je infracrvenom (FTIR) i refleksionom spektroskopijom. FTIR spektri su identifikovali C=O grupe flavonoida, O-H, C-O i C-O-C grupe fenola, potvrđujući prisustvo liganada na površini nanočestica TiO2 . Kubelka-Munk transformacijom spektara difuzione refleksije vidi se pomeraj ekscitacije /DHQ prema vidljivom delu spektra. Ispitivanje citotoksičnosti MTT testom urađeno je na zdravim ljudskim MRC-5 ćelijama, kao i na ljudskim HeLa ćelijama raka grlića materice. Takođe, H2DCFDA testom ispitan je efekat TiO2 /DHQ na proizvodnju reaktivnih vrsta kiseonika u MRC-5 ćelijama.This study aims to modify the surface of TiO2 nanoparticles with bioactive ligands, forming an interfacial charge transfer complex to achieve its activation under visible light. Dihydroquercetin (DHQ) is a catechol-type ligand with pronounced antioxidant and valuable biological performance. The inorganic-organic hybrid nanocomposite TiO2 /DHQ was characterized by Fourier transform infrared (FTIR) and Reflectance spectroscopy. FTIR spectra identified C=O stretching in flavones, O-H, C-O and C-O-C starching in phenolic compounds, demonstrating the presence of ligands on the surface of TiO2 nanoparticles. The Kubelka-Munk transformation of the diffuse reflection spectra shows a shift in the excitation of the TiO2 /DHQ towards the visible part of the spectrum. Cytotoxicity testing was performed on healthy human MRC-5 cells and human cervical cancer HeLa cells determined by MTT assay. Also, the effect of TiO2 /DHQ on the production of reactive oxygen species in MRC-5 cells was determined by the H2DCFDA assay.59th Meeting of the Serbian Chemical Society; June 1-2, 2023, Novi Sad, Serbi

Prospective study of cytotoxic and genotoxic effects of Combretastatin

Combretastatins are a class of natural phenols found in the bark of Combretum caffrum, commonly known as South African Bush Willow. Despite having a similar name, combretastatins are unrelated to statins, a family of cholesterol-lowering drugs. Combretastatin A4 have been shown to be one of the most potent tubulin-depolymerizing agent. Microtubules control chromosomal segregation and cytokinesis during mitosis in both cancer and stromal cells and contribute to overall tumor growth. Consequently, microtubule inhibitors interfere with cell cycle progression and induce apoptosis in cancer cells in vitro. The aim of this study was to investigate the potential gentoxic effect of Comretastatin A4 (CA4) in isolated peripheral blood mononuclear cells (PBMC) in Comet assay in order to establish is there any DNA damage in healty non-dividing cells. The aim also was to explore potential cytotoxic activity of CA4 against human cervical carcinoma (HeLa) cell line. Genotoxicity of CA4 was evaluated on PBMC in a range of 9 concentrations (from 1 nM to 200μM). Non of the tested concentrations showed genotoxiceffect. The same range of different concentrations of CA4 (from 1 nM to 200μM) were applied to evaluate potential cytotoxicity in a monolayer culture of HeLa cells using the 3-[4,5-dimethylthiazole-2-yl]-2,5-diphenyltetrazolium bromide (MTT) assay. After 24h incubation with CA4, there was a significant reduction in cell viability in all concentrations above 250 nm, while IC50 (half maximal inhibitory concentration) was 123 ± 0.06396 μM. We concluded that CA4 does not have gentoxic effect on PBMC, and that it reduce cell viability of cancer HeLa cell lines. These results are especialy importanat because they showed that CA4 does not damage the DNA molecule in healthy human cells, but achieves its cytotoxic effect on malignant cells in the same range of concentrations.51st European Environmental Mutagenesis and Genomics Society (EEMGS) & 27th Spanish Environmental Mutagenesis and Genomics Society (SEMA) meeting,May 15th-18th, 2023 Málaga (Spain

Dry Olive Leaf Extract Counteracts L-Thyroxine-Induced Genotoxicity in Human Peripheral Blood Leukocytes In Vitro

The thyroid hormones change the rate of basal metabolism, modulating the consumption of oxygen and causing production of reactive oxygen species, which leads to the development of oxidative stress and DNA strand breaks. Olive (Olea europaea L.) leaf contains many potentially bioactive compounds, making it one of the most potent natural antioxidants. The objective of this study was to evaluate the genotoxicity of L-thyroxine and to investigate antioxidative and antigenotoxic potential of the standardized oleuropein-rich dry olive leaf extract (DOLE) against hydrogen peroxide and L-thyroxine-induced DNA damage in human peripheral blood leukocytes by using the comet assay. Various concentrations of the extract were tested with both DNA damage inducers, under two different experimental conditions, pretreatment and posttreatment. Results indicate that L-thyroxine exhibited genotoxic effect and that DOLE displayed protective effect against thyroxine-induced genotoxicity. The number of cells with DNA damage, was significantly reduced, in both pretreated and posttreated samples (P lt 0.05). Comparing the beneficial effect of all tested concentrations of DOLE, in both experimental protocols, it appears that extract was more effective in reducing DNA damage in the pretreatment, exhibiting protective role against L-thyroxine effect. This feature of DOLE can be explained by its capacity to act as potent free radical scavenger

Genotoksični potencijal nesteroidnih hormona

Hormones are cellular products involved in the regulation of a large number of processes in living systems, and which by their actions affect the growth, function and metabo­lism of cells. Considering that hormones are compounds normally present in the organism, it is important to determine if they can, under certain circumstances, lead to genetic changes in the hereditary material. Numerous experimental studies in vitro and in vivo in different systems, from bacteria to mammals, dealt with the mutagenic and genotoxic effects of hormones. This work presents an overview of the research on genotoxic effects of non­steroidal hormones, although possible changes of genetic material under their influence have not still been known enough, and moreover, investigations on their genotoxic influ­ence have given conflicting results. The study results show that mechanisms of genotoxic effect of nonsteroidal hormones are manifested through the increase of oxidative stress by arising reactive oxygen species. A common mechanism of ROS occurence in thyroid hormones and catecholamines is through metabolic oxidation of their phenolic groups. Mani­festation of insulin genotoxic effect is based on production of ROS by activation of NADPH isophorms, while testing oxytocin showed absence of genotoxic effect. Considering that the investigations on genotoxicity of nonsteroidal hormones demonstrated both positive and negative results, the explanation of this discordance involve limitations of test systems themselves, different cell types or biological species used in the experiments, different lev­el of reactivity in vitro and in vivo, as well as possible variations in a tissue-specific expres­sion. Integrated, the provided data contribute to better understanding of genotoxic effect of nonsteroidal hormones and point out to the role and mode of action of these hormones in the process of occurring of effects caused by oxidative stress.Hormoni su ćelijski proizvodi uključeni u regulaciju velikog broja procesa u živim sistemima koji svojim dejstvom utiču na rast, funkciju ili metabolizam ćelija. Obzirom da su hormoni jedinjenja koja su uobičajeno prisutna u organizmu, značajno je utvrditi da li oni mogu pod izvesnim okolnostima dovesti do genetičkih promena na naslednom materijalu. Eksperimentalna ispitivanja u in vitro i in vivo uslovima u različitim sistemima, od bakterija do sisara, bavila su se istraživanjem mutagenih i genotoksičnih efekata hormona. U ovom radu je dat pregled istraživanja genotoksičnih efekata nesteroidnih hormona, pošto još uvek nisu dovoljno poznate moguće promene naslednog materijala pod njihovim uticajem, a i ispitivanja njihovog genotoksičnog dejstva su dala oprečne rezultate. Rezultati studija pokazuju da se mehanizmi genotoksičnog dejstva nesteroidnih hormona ispoljavaju kroz povećanje oksidativnog stresa nastankom reaktivnih vrsta kiseonika (reactive oxygen species - ROS). Uobičajeni mehanizam nastanka ROS kod tireoidnih hormona i kateholamina je putem metaboličke oksidacije njihovih fenolnih grupa. Ispoljavanje genotoksičnog efekta insulina se zasniva na produkciji ROS putem aktivacije NADPH izoformi, dok je ispitivanje oksitocina pokazalo odsustvo genotoksičnog efekta. Uzimajući u obzir da su ispitivanja genotoksičnosti nesteroidnih hormona pokazala i pozitivne i negativne rezultate, objašnjenja ovog neslaganja obuhvataju ograničenja samih test sistema, različite tipove ćelije ili bioloških vrsta upotrebljenih u eksperimentima, različiti nivo reaktivnosti u in vitro i in vivo uslovima, kao i moguće razlike u tkivno specifičnoj ekspresiji. Objedinjeni, navedeni podaci doprinose boljem razumevanju genotoksičnih efekata nesteroidnih hormona i ukazuju na ulogu i načine delovanja ovih hormona u procesu nastanka efekata izazvanih oksidativnim stresom

Evaluacija antigenotoksičnog potencijala salvianolične kiseline B u prisustvu vodonik-peroksida na leukocitima periferne krvi in vitro

Aim. Oxidative stress is a consequence of the increased production of free radicals that is caused by the disturbance in the balance of oxidation-reduction activity. Salvianolic acid B is a polyphenol compound, derived from the plant Salvia miltiorrhiza Bunge, which showed significant antioxidant properties. The aim of this study is the investigation of genotoxic potential of salvianolic acid B and evaluation of its antigenotoxic activity against the DNA damage induced by hydrogen peroxide in peripheral blood leukocytes in vitro, using the alkaline Comet assay. Materials and Methods. The evaluation of the ability of various concentrations of salvianolic acid B (12.5μM, 25μM and 50 μM) to reduce the number of cells with DNA damage caused by hydrogen peroxide as an oxidant was performed under two experimental protocols: pretreatment and cotreatment, in order to determine antigenotoxicity on preventive and intervention levels. Results. Results indicate that the salvianolic acid B did not exhibit a genotoxic effect after 30 minutes of incubation, in the tested concentrations. In the pretreatment, a concentration of 50 μM showed a significant decrease of the hydrogen peroxide induced DNA damage. Salvianolic acid B was more effective in reducing DNA damage in cotreatment, where concentrations of 25 μM and 50 μM demonstrated a significant abrogation of DNA damage. Protective effect of salvianolic acid B was dependent on the concentration. Conclusions. The results showed that salvianolic acid B has pronounced antigenotoxic effect on the intervention level, which makes it a potential agent in treatment of diseases in which oxidative DNA damage plays an important role.Cilj. Oksidativni stres je posledica prekomerne produkcije slobodnih radikala i nastaje usled poremećaja u ravnoteži oksido-redukcionih procesa. Salvianolična kiselina B je polifenolno jedinjenje, poreklom iz biljke Salvia miltiorrhiza Bunge, koje je pokazalo značajna antioksidativna svojstva. Cilj rada bio je da se ispita genotoksični potencijal salvianolične kiseline B i izvrši evaluacija njene antigenotoksične aktivnosti na DNK oštećenja indukovana vodonik-peroksidom u leukocitima periferne krvi in vitro, primenom alkalnog Komet testa. Materijal i metode. Procenjena je sposobnost različitih koncentracija salvianolične kiseline B (12,5 μM, 25 μM i 50 μM) da redukuju broj ćelija sa DNK oštećenjem izazvanim vodonik-peroksidom kao oksidansom u okviru dva eksperimentalna protokola: pretretmana i kotretmana radi utvrđivanja antigenotoksičnosti na preventivnom i interventnom nivou. Rezultati. Rezultati su pokazali da salvianolična kiselina B, nakon 30 minuta inkubacije, nije ispoljila genotoksičan efekat u ispitivanim koncentracijama. U pretretmanu, koncentracija od 50 μM pokazala je značajnu redukciju DNK oštećenja indukovanih vodonik-peroksidom. Salvianolična kiselina B bila je efikasnija u redukciji DNK oštećenja u kotretmanu, gde su koncentracije od 25 μM i 50 μM pokazale značajan efekat redukcije nivoa DNK oštećenja. Pokazani protektivni efekat salvianolične kiseline B zavisio je od koncentracije. Zaključak. Dobijeni rezultati pokazali su da salvianolična kiselina B ima izraženiji antigenotoksični efekat na interventnom nivou, što je čini potencijalnim agensom za primenu kod oboljenja u kojima oksidativna DNK oštećenja imaju važnu ulogu

The effect of influenza vaccine immunization on natural antibodies

Natural, polyreactive, low-affinity antibodies are known to play an important role not only in the immediate defense against pathogens, but also in shaping the acquired immune response. On the other hand, antigen specific, high-affinity antibodies can affect the balance of natural antibodies and lead to autoimmune diseases. In this study we have analyzed the changes that occur in the IgM and IgG pool of natural antibodies after immunization with split or whole virion influenza vaccine. For this purpose, “in-house” developed ELISAs were used. The subjects were divided, according to the vaccination status, into those who had been immunized with the influenza vaccine in previous years and those who had been immunized for the first time. The analysis indicated that the pool of natural antibodies was not impaired by the immunization, evidenced by the lack of changes in any of the groups, and that certain fluctuations were induced in order to maintain the homeostasis of the immune system

Antigenotoxic Properties of Agaricus blazei against Hydrogen Peroxide in Human Peripheral Blood Cells

The ability of Agaricus blazei mushroom in its dried and powdered mycelial form was evaluated for its antigenotoxic properties for the first time. Antigenotoxic effects in human peripheral blood cells against H2O2-induced DNA damage were examined in pretreatment and posttreatment protocol by comet assay. The results showed better antigenotoxic properties of Agaricus blazei on the interventional level, respectively, after treatment. Agaricus blazei in concentration of 250 mu g/mL after treatment was most efficient in regard to its action against DNA damage. The evaluation of repair kinetics showed decrease in H2O2 induced DNA damage 15min after the application of A. blazei, reaching the maximum potency after 30 min. Analysis of antioxidant properties of Agaricus blazei revealed strong center dot OH scavenging properties and moderate reducing power, while its DPPH scavenging ability was weak. In regard to our findings, we can conclude that our preliminary results demonstrated antigenotoxic properties of Agaricus blazei and its strong center dot OH scavenging ability. Mechanisms underlying its properties should be further evaluated in in vivo studies

Evaluation of genotoxic and antigenotoxic properties of essential oils of Seseli rigidum Waldst. & Kit. (Apiaceae)

The essential oils of genus Seseli are known for their beneficial biological activities and could present novel targets in the development of safe and effective preparations of plant products. The objective was to test the essential oils of different parts of Seseli rigidum from two natural habitats for potential genotoxic and antigenotoxic activities against H2O2-induced DNA damage in human whole blood cells in vitro, by the comet assay. The essential oil analysis showed a high falcarinol content in oil from the root, while oils of the fruit and aerial parts contained a-pinene as the main compound. Genotoxicity was not detected at any of the concentrations of the essential oils from the three parts of the plant from localities I and II. Although the antioxidant activity (established by the FRAP and DPPH tests) of the investigated oils was low, all oils demonstrated a strong antigenotoxic effect against H2O2-induced damage post-treatment, when the oils were applied after the oxidant. Based on the lack of pretreatment activity and the post-treatment reduction in DNA damage, the antigenotoxic effect of S. rigidum essential oils was probably based on the stimulation of DNA repair mechanisms. Environmental conditions did not affect the antigenotoxic properties of the oils. In conclusion, our results revealed the antigenotoxic properties of S. rigidum essential oils and appropriate and safe doses with beneficial effects under the described conditions

Cytogenetic alterations in rheumatoid arthritis patients treated with methotrexate and dry olive leaf extract

Oxidative stress and inflammation are DNA instability factors for rheumatoid arthritis (RA) patients. The aims of this study were to evaluate cytogenetic alterations in Peripheral Blood Lymphocytes (PBL) in two groups of RA patients: the early and the long-term RA group; and to examine potential of concomitant treatment with Methotrexate (MTX) and Dry olive leaf extract (DOLE) against cytogenetic damage in RA patients after a 3-weeks treatment. A total of 32 RA patients and 10 healthy individuals were included. RA patients were equally divided into four groups: two groups with early phase RA (one treated with MTX alone, the other in combination with DOLE); and two long-term phase RA groups (group with active disease and group with low disease activity)-both treated with MTX and DOLE combination. PBL cultures were screened for chromosome aberrations and micronuclei frequencies. Significantly increased frequencies of micronuclei were shown in active phase RA disease (both early and long-term) but not in the group with low disease activity, as compared to controls. Chromosome aberrations were detected for all 4 RA groups. The highest frequencies of micronuclei and chromosome aberrations were found in the long-term active RA group. After 3 weeks-treatment, there were no significant decrease of the micronuclei frequencies compared to baseline, although they were reduced in all RA groups, except for the group with the long-term active disease. High level of cytogenetic damage in RA patients was concordant with duration and activity of the RA disease. At 3 weeks of therapy, neither the combined treatment (MTX+DOLE), nor MTX alone did not affect the frequency of micronuclei formation

Toxicity of Silver Nanoparticles Supported by Surface-Modified Zirconium Dioxide with Dihydroquercetin

The antibacterial performance and cytotoxic examination of in situ prepared silver nanoparticles (Ag NPs), on inorganic-organic hybrid nanopowder consisting of zirconium dioxide nanoparticles (ZrO2 NPs) and dihydroquercetin (DHQ), was performed against Gram (−) bacteria Escherichia coli and Gram (+) bacteria Staphylococcus aureus, as well as against human cervical cancer cells HeLa and healthy MRC-5 human cells. The surface modification of ZrO2 NPs, synthesized by the sol-gel method, with DHQ leads to the interfacial charge transfer (ICT) complex formation indicated by the appearance of absorption in the visible spectral range. The prepared samples were thoroughly characterized (TEM, XRD, reflection spectroscopy), and, in addition, the spectroscopic observations are supported by the density functional theory (DFT) calculations using a cluster model. The concentration- and time-dependent antibacterial tests indicated a complete reduction of bacterial species, E. coli and S. aureus, for all investigated concentrations of silver (0.10, 0.25, and 0.50 mg/mL) after 24 h of contact. On the other side, the functionalized ZrO2 NPs with DHQ, before and after deposition of Ag NPs, do not display a significant decrease in the viability of HeLa MRC-5 cells in any of the used concentrations compared to the contro