Effects of ethanolic olive leaf extract (Olea europaea L.) on genomic instability, parameters of oxidative stress and inflammation in rheumatoid arthritis patients

Reumatoidni artritis (RA) predstavlja autoimunsko inflamatorno oboljenje, u čijem razvoju ključnu ulogu imaju inflamatorni medijatori i oksidativni stres, koji mogu dovesti do oštećenja biomolekula i nastanka genomske nestabilnosti. Suvi etanolni ekstrakt lista masline (engl. dry olive leaf extract, DOLE) je poznat po izuzetnim antioksidativnim, antiinflamatornim i genoprotektivnim dejstvima. Međutim, do sada nisu ispitivani njegovi efekti u terapiji RA. Cilj istraživanja ove doktorske disertacije je poređenje efekata tronedeljne i šestonedeljne kombinovane terapije metotreksatom i DOLE u odnosu na efekte standardne terapije samo metotreksatom, kod pacijenata sa RA. U istraživanju je učestvovalo 8 pacijenata novodijagnostikovanih za RA, 16 pacijenata sa dugotrajnim RA, koji su bili na kombinovanoj terapiji, dok je posebnu grupu činilo 8 novodijagnostikovanih pacijenata na terapiji samo metotreksatom. Kod sve tri grupe, pre uvođenja eksperimentalne terapije, zabeležen je povišen nivo inflamacije, oksidativnih oštećenja lipida, proteina i DNK, kao i povećana učestalost mikronukleusa u odnosu na vrednosti kod zdravih kontrolnih ispitanika. Rezultati tronedeljne i šestonedeljne terapije su pokazali da kombinovana primena DOLE i metotreksata daje bolje efekte u odnosu na terapiju samo metotreksatom, pre svega u pogledu efikasnije redukcije primarnih DNK oštećenja, kao i redukcije nivoa interleukina-6, kod pacijenata u ranoj fazi RA. Kombinovana terapija je takođe pokazala efekat u redukciji nitrita kod grupe u ranoj fazi RA i smanjenje lipidne peroksidacije kod dugotrajno obolelih pacijenata. Praćenjem učestalosti mikronukleusa u limfocitima, nisu utvrđeni značajni efekti terapije na nivo genomske nestabilnosti posle šest nedelja.Pokazani su pozitivni efekti kombinovane terapijske primene metotreksata i DOLE na oksidativna oštećenja ćelija kod pacijenata u ranoj fazi RA, u poređenju sa terapijom samo metotreksatom. Kombinovana terapija je bila efikasnija u ranoj fazi RA, dok su njeni efekti slabije izraženi kod dugotrajnog stanja aktivne bolesti

Alterations of acrocentric chromosomes in peripheral blood lymphocytes in patients with Alzheimer's disease

While Alzheimer's disease (AD) is primarily considered to be a neurodegenerative disorder, there is much evidence supporting the idea that it is also a systematic disease, so the occurrence of genomic instability in cells that are not neurons is equally important. Accordingly, acrocentric chromosomes play an important role in the etiology of AD, especially the presence of trisomy 21. The aim of this work was to assess the relationship between the frequency of acrocentrics included in satellite association (SA) and acrocentrics with premature centromere division (PCD) in the peripheral blood lymphocytes of AD patients and age-matched controls. Since our results showed that there are significant differences in SA frequency, as well as in the frequency of PCD in acrocentrics between AD patients and control group, we may conclude that the occurrence of acrocentric chromosome alterations presented in this study could be related to the etiology of AD

Unexpected effect of dry olive leaf extract (DOLE) before and after CaNa2EDTA chelation therapy in comet assay in lead intoxicated workers

The CaNa2EDTA chelation is a standard therapy for lead (Pb) intoxication in occupationally exposed workers. Application of antioxidant nutrients through exogenous supplementation is often practiced with the chelation therapy, although their synergistic effect in reduction of Pb-induced oxidative damage has not been investigated conclusively. Dry olive Leaf extract (DOLE) is polyphenol rich natural antioxidant. The effects of DOLE on the levels of DNA damage were investigated ex vivo in peripheral blood lymphocytes (PBLs) of 19 male workers occupationally exposed to Pb, before and after application of five day CaNa2EDTA chelation therapy. Comet assay was used to assess levels of DNA damage. While the level of DNA damage in PBLs of workers before chelation were moderately increased (24.21±14.26) compared to controls (6.0±3.37), the incubation of the same samples with 1mg/mL DOLE for 30 min at 370C lead to a severe increase in DNA damage levels (64.03±20.96). After the exposure of workers to a five day CaNa2EDTA chelation regimen, the experiment was repeated. Following chelation, the level of DNA damage in PBLs of workers was decreased (8.26±4.62) significantly compared to the baseline value and was then similar to the control level. When the PBLs after chelation were treated with 1mg/mL DOLE for 30 min, high level of damage was obtained (41.82±23.17). The antigenotoxic effects of five day CaNa2EDTA chelation were demonstrated in PBLs of Pb exposed workers. On the contrary, the applications of DOLE lead to an increase of oxidative DNA damage after 30 min incubation, exhibiting prooxidant rather than antioxidant effect. After CaNa2EDTA treatment, the acute prooxidant effects of DOLE remained following the incubation, but, the oxidative DNA damage was less severe compared to the same experiment with DOLE before the chelation, probably as a result of partial removal of Pb from cells by chelation therapy. Prooxidant nature of DOLE could be a result of Pb-mediated hydroxyl radical formation, where heavy metals serve as catalysts for the reactions which oxidize DOLE and reduce oxygen. Removal of Pb by complexation with CaNa2EDTA seems to significantly depress these oxidative events. However, this mechanism remains to be explored on molecular level. It could be concluded that the DOLE exhibits prooxidant effects in presence of Pb in lymphocytes of exposed workers, and its effect is less pronounced following the removal of Pb after standard chelation therapy.3rd International Congress on Occupational & Environmental Toxicology - ICOETox 2016 and 3rd Ibero-American Meeting on Toxicology and Environmental Health International (IBAMTOX 2016), 21-23 June 2016 | Port

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

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

Evaluacija oštećenja DNK u limfocitima mladih, starih i pacijenata obolelih od Alchajmerove bolesti tretiranih β-estradiolom u komet testu

Background: The antioxidant activity of estrogen has a beneficial impact in Alzheimer's disease. A variety of clinical studies have demonstrated that estrogen replacement therapy in postmenopausal women results in a lower frequency of AD, delaying the onset of the neurodegenerative cascade. On the other hand, it has been demonstrated that estrogens may exhibit genotoxic effects, especially at elevated tissue concentrations. Therefore, the aim of this study was to determine whether β-estradiol induces DNA damage in the peripheral blood lymphocytes of healthy young females and males, healthy elderly females and males and females and males with Alzheimer's disease. Methods: All experiments were performed using the alkaline version of the Comet assay (single cell gel electrophoresis), on six donors per each experimental group and controls. Results: In the Comet assay, a significant increase of DNA migration was observed in the lymphocytes of all treated groups (young and elderly females, young and elderly males, AD females and AD males) at all β-estradiol concentrations (50 µmol/L, 100 µmol/L and 250 µmol/L) used in this investigation. In all the experiments cell viability was over 80%.Conclusions: Lymphocytes are sensitive to the test concentrations of β-estradiol in the Comet assay regardless of gender, age and health condition of the examined subjects. Therefore, the role of β-estradiol in cellular DNA damage has been confirmed.Uvod: Antioksidativna svojstva estrogena imaju povoljan uticaj na Alchajmerovu bolest (AB). Brojne kliničke studije su pokazale da primena hormonske supstitucione terapije estrogenom kod žena u postmenopauzi smanjuje učestalost pojave AB, odlažući početak neurodegenerativnih procesa. S druge strane, pokazano je da estrogeni mogu ispoljiti genotoksičan efekat, naročito pri povišenim koncentracijama u tkivu. Shodno tome, cilj ove studije bio je ispitivanje sposobnosti β-estradiola da izazove oštećenja u limfocitima periferne krvi zdravih mladih žena i muškaraca, zdravih starih žena i muškaraca, kao i žena i muškaraca koji boluju od Alchajmerove bolesti. Metode: Svi eksperimenti su izvedeni primenom alkalne verzije komet testa (gel-elektroforeza DNK pojedinačnih ćelija), na po šest subjekata u svakoj ispitivanoj grupi. Rezultati: Upotrebom komet testa, zabeleženo je značajno povećanje migracije DNK u limfocitima ispitanika iz svih tretiranih grupa (mladih i starih žena, mladih i starih muškaraca kao i kod žena i muškaraca sa AB), pri svim koncentracijama β-estradiola (50 µmol/L, 100 µmol/L i 250 µmol/L) koje su korišćene u ovoj studiji. U svim eksperimentima vijabilnost ćelija je bila iznad 80%. Zaključak: Limfociti periferne krvi osetljivi su na testirane koncentracije β-estradiola, bez obzira na pol, godište i zdravstveno stanje ispitanika. U skladu s navedenim nalazima, potvrđena je uloga β-estradiola u izazivanju oštećenja na ćelijskoj DNK

DOLE protects leucocytes from hormone-induced DNA damage in vitro

Both hormones thyroxin and adrenaline increase endogenous formation of reactive oxygen species (ROS), leading to oxidative stress and DNA damage. Dry olive leaf extract (DOLE) is plant product known to modulate effects of oxidants in human cells. Aim of our study was to evaluate ability of commercial DOLE to protect nuclear DNA from oxidative damage induced by different oxidants and to compare level of protective effect by using comet assay. Peripheral blood leukocytes were treated in vitro with DOLE under two protocols. First group of samples was exposed to oxidant and afterwards with three concentrations of DOLE (0,125, 0,5 and 1 mg/ml). Second group was pretreated with DOLE under same conditions, followed by the administration of oxidant. Effect of the extract with adrenaline treatment was most beneficial at smallest concentrations (0,125 mg/ml), while in treatment with thyroxin DOLE was most effective at 1 mg/ml in pretreatment and at 0.5 mg/ml in posttreatment. DOLEs protective effect was noticeable under both protocols compared to only oxidant exposed controls. Using the comet assay methodology, we were able to detect antioxidant and genoprotective properties of DOLE

Effects of ethanolic olive leaf extract (Olea europaea L.) on genomic instability, parameters of oxidative stress and inflammation in rheumatoid arthritis patients

Reumatoidni artritis (RA) predstavlja autoimunsko inflamatorno oboljenje, u čijem razvoju ključnu ulogu imaju inflamatorni medijatori i oksidativni stres, koji mogu dovesti do oštećenja biomolekula i nastanka genomske nestabilnosti. Suvi etanolni ekstrakt lista masline (engl. dry olive leaf extract, DOLE) je poznat po izuzetnim antioksidativnim, antiinflamatornim i genoprotektivnim dejstvima. Međutim, do sada nisu ispitivani njegovi efekti u terapiji RA. Cilj istraživanja ove doktorske disertacije je poređenje efekata tronedeljne i šestonedeljne kombinovane terapije metotreksatom i DOLE u odnosu na efekte standardne terapije samo metotreksatom, kod pacijenata sa RA. U istraživanju je učestvovalo 8 pacijenata novodijagnostikovanih za RA, 16 pacijenata sa dugotrajnim RA, koji su bili na kombinovanoj terapiji, dok je posebnu grupu činilo 8 novodijagnostikovanih pacijenata na terapiji samo metotreksatom. Kod sve tri grupe, pre uvođenja eksperimentalne terapije, zabeležen je povišen nivo inflamacije, oksidativnih oštećenja lipida, proteina i DNK, kao i povećana učestalost mikronukleusa u odnosu na vrednosti kod zdravih kontrolnih ispitanika. Rezultati tronedeljne i šestonedeljne terapije su pokazali da kombinovana primena DOLE i metotreksata daje bolje efekte u odnosu na terapiju samo metotreksatom, pre svega u pogledu efikasnije redukcije primarnih DNK oštećenja, kao i redukcije nivoa interleukina-6, kod pacijenata u ranoj fazi RA. Kombinovana terapija je takođe pokazala efekat u redukciji nitrita kod grupe u ranoj fazi RA i smanjenje lipidne peroksidacije kod dugotrajno obolelih pacijenata. Praćenjem učestalosti mikronukleusa u limfocitima, nisu utvrđeni značajni efekti terapije na nivo genomske nestabilnosti posle šest nedelja.Pokazani su pozitivni efekti kombinovane terapijske primene metotreksata i DOLE na oksidativna oštećenja ćelija kod pacijenata u ranoj fazi RA, u poređenju sa terapijom samo metotreksatom. Kombinovana terapija je bila efikasnija u ranoj fazi RA, dok su njeni efekti slabije izraženi kod dugotrajnog stanja aktivne bolesti

EDTA helatna terapija smanjuje oksidativno DNK oštećenje u limfocitima periferne krvi radnika profesionalno izloženih olovu

INTRODUCTION: Pathogenetic effect of lead (Pb) is multifactorial and it is known that one of the ways which lead damages cellular components is by direct formation of reactive oxygen species. ...VIII simpozijum o hemijskim štetnostima na radnom mestu - uticaj na zdravlje radnika i stanovništva (sa međunarodnim učešćem), Zlatibor, 26-28, septembar 2012. godin

Biomarkers of Oxidative Stress in Workers Exposed To Lead

One of the numerous adverse manifestations following lead (Pb) exposure is production of free radicals and increased oxidative stress, which can lead to altered enyzme functioning and degradation of structural components in organism. The objective was to investigate the effects of Pb exposure on biomarkers of oxidative stress and antioxidant defence system in 15 adult male workers after occupational exposure to lead versus unexposed healthy controls. The impact of Pb on oxidative stress parameters was evaluated by measuring blood and urinary Pb concentrations, plasma levels of antioxidant enzymes catalase (CAT), superoxide dismutase (SOD), level of malondialdehyde (MDA) as the lipid peroxidation indicator, concentrations of carbonyl groups, plasma protein thiols and nitrites as biomarkers of protein oxidative modification. The results of MDA concentration revealed increased lipid peroxidation (P < 0.001), while the rise of protein carbonyl and nitrite levels (P < 0.001, P < 0.006, respectively) and decrease of plasma thiols (P < 0.001) indicated significant protein damage in Pb exposed workers, compared to unexposed controls. Increased activity of antioxidant enzymes SOD and CAT (P < 0.001) was also determined in exposed group. Elevated blood lead levels showed strong positive correlation (r=0.809, P < 0.001) with increased CAT concentration. Our findings indicate that Pb promotes oxidative damage in exposed subjects, and that increased lead content can affect the activity of antioxidant enzymes. Considering that oxidative stress has been recognized in etiology of many diseases, future explorations on impact of these changes in populations occupationally exposed to lead are suggested