Evaluation of antigenotoxic potential of ethanolic olive leaf extract (Olea europaea L.) on the effect of thyroxine, adrenaline, estradiol and diethylstilbestrol in human peripheral blood leukocytes in vitro

Hormoni su organska jedinjenja različite hemijske prirode koji svojim dejstvom utiču na rast, funkciju i metabolizam organizma. Povišene koncentracije hormona dovode do stvaranja slobodnih radikala, što može izazvati oksidativni stres i oštećenja DNK molekula. Poznato je da komponente tradicionalne mediteranske ishrane imaju pozitivne efekte na smanjenje oksidativnog stresa i prevenciju mnogih bolesti. Brojne in vitro i in vivo studije su potvrdile korisne efekte suvog ekstrakta lista masline (DOLE) i njegovih sastojaka, kao i zaštitni potencijal u odnosu na oksidativna oštećenja DNK molekula. Stoga, glavni cilj ove doktorske disertacije je bio da se ispita antigenotoksični potencijal spektra koncentracija suvog ekstrakta lista masline u leukocitima periferne krvi čoveka u prisustvu hormona tiroksina, adrenalina, estradiola i dietilstilbestrola in vitro. Ispitivanje je obavljeno primenom alkalnog komet testa, osetljive i brze metode za određivanje i analizu primarnih oštećenja DNK u pojedinačnim ćelijama. Rezultati ove studije su pokazali da su tiroksin, adrenalin, estradiol i dietilstibestrol sposobni da budu medijatori značajnog povećanja oštećenja DNK molekula. Suvi ekstrakt lista masline je u svim testiranim koncentracijama ispoljio značajan antigenotoksični potencijal u oba eksperimentalna protokola, u pretretmanu i posttretmanu. Praćenje kinetike reparacije DNK u prisustvu DOLE u odnosu na oštećenja izazvana hormonima je pokazalo da ekstrakt nije značajno uticao na stimulaciju reparacije oštećenja DNK. Sumirajući rezultate ove disertacije, može se zaključiti da suvi ekstrakt lista masline poseduje izražen potencijal smanjenja primarnih oštećenja DNK izazvanih tiroksinom, adrenalinom, estradiolom i dietilstilbestrolom. Predstavljeni in vitro model na leukocitima periferne krvi čoveka pružio je podatke koji su korisni za buduće in vivo studije i klinička ispitivanja suvog ekstrakta lista masline.Hormones are organic compounds of different chemical nature that affect cell growth, function and metabolism. Elevated concentrations of hormones lead to the formation of free radicals, which can cause oxidative stress and damage to the DNA molecule. It is known that the components of traditional Mediterranean food have positive effects on the reduction of oxidative stress and the prevention of many diseases. Numerous in vitro and in vivo studies have confirmed the beneficial effects of dry olive leaf extract (DOLE) and its constituents, as well as protective potential on the oxidative damage of DNA molecules. Therefore, the main aim of this doctoral dissertation was to investigate the antigenotoxic potential of the spectrum of concentrations of dry olive leaf extract in peripheral blood leukocytes in the presence of hormone thyroxine, adrenaline, estradiol, and diethylstilbestrol in vitro. The study was performed using an alkaline comet assay, a sensitive and fast method for determining and analyzing primary DNA damage in individual cells. The results of this study have shown that thyroxine, adrenaline, estradiol, and diethylstibestrol are capable of being mediators of significant increase of DNA damage. Dry olive leaf extract demonstrated significant ability to reduce primary DNA damage at all tested concentrations and in both experimental protocols, in pretreatment and posttreatment. Monitoring the kinetics of DNA repair in the presence of DOLE in relation to damage induced by homones has shown that the extract did not significantly affect the stimulation of DNA damage repair. Summarizing the results of this dissertation, it can be concluded that the dry olive leaf extract possesses a strong potential to reduce primary DNA damage induced by thyroxine, adrenaline, estradiol, and diethylstilbestrol. The presented in vitro model on human peripheral blood leukocytes provided data that are useful for future in vivo studies and clinical trials of dry olive leaf extract

Antigenotoxic Effects of Biochaga and Dihydroquercetin (Taxifolin) on H2O2-Induced DNA Damage in Human Whole Blood Cells

The health benefits of natural products have long been recognized. Consumption of dietary compounds such as supplements provides an alternative source of natural products to those obtained from the diet. There is a growing concern regarding the possible side effects of using different food supplements simultaneously, since their possible interactions are less known. For the first time, we have tested genotoxic and antigenotoxic effects of Biochaga, in combination with dihydroquercetin. No genotoxic effect on whole blood cells was observed within individual treatment of Biochaga (250 μg/mL, 500 μg/mL and 1000 μg/mL) and dihydroquercetin (100 μg/mL, 250 μg/mL and 500 μg/mL), nor in combination. Afterwards, antigenotoxic potency of both supplements against hydrogen peroxide- (H2O2-) induced DNA damage to whole blood cells (WBC) was assessed, using the comet assay. Biochaga and dihydroquercetin displayed a strong potential to attenuate H2O2-induced damage on DNA in cells at all tested concentrations, with a statistical significance (p < 0:05), whereas Biochaga at the dose of 500 μg/mL in combination with dihydroquercetin 500 μg/mL was most prominent. Biochaga in combination with dihydroquercetin is able to protect genomic material from oxidative damage induced by hydrogen peroxide in vitro

Analiza DNK oštećenja izazvanog tiazofurinom u humanim ćelijama pune krvi primenom in vitro komet testa

Objective. Inosine 5’-monophosphate dehydrogenase (IMPDH) activity in cancer cells is increased. Tiazofurin selectively inhibits the activity of IMPDH, and it has been granted for the treatment of different cancers and new viral diseases. Its widespread use was limited because exposure to tiazofurin under certain circumstances was found to have a higher frequency of severe non-hematologic toxicity. Therefore, the objective of this study was to examine genotoxic action and inducement of DNA damage of tiazofurin using the comet assay. Methods. The ability of tiazofurin to induce DNA damage was evaluated using single-cell gel electrophoresis (SCGE) technique/comet assay. Human whole blood cells were exposed to three final concentrations of tiazofurin (1 µM/mL, 2 µM/mL, and 5 µM/mL) for 30 min in vitro. Results. Our results indicate that tiazofurin produced a significant level of DNA damage on whole blood cells after 30 min of exposure vs. control. All tested concentrations were significantly comet-forming, in a concentration-dependent manner. Conclusion. Our investigation on the tiazofurin-treated cells and their relationship to the formation of DNA damage demonstrated that the genotoxic effect was induced after exposure to tiazofurin under described conditions.Cilj. Aktivnost inozin 5'-monofosfat dehidrogenaze (IMPDH) povec'ana je u c'elijama karcinoma. Tiazofurin selektivno inhibira aktivnost IMPDH i odobren je za lečenje različitih karcinoma i novih virusnih bolesti. Njegova široko rasprostranjena upotreba bila je ograničena jer je utvrđeno da je izloženost tiazofurinu pod određenim okolnostima imala vec'u incidencu ozbiljne nehematološke toksičnosti. Stoga je cilj ove studije bio da se pomoc'u komet testa ispita genotoksično delovanje i izazivanje DNK oštec'enja tiazofurinom. Metode. Sposobnost tiazofurina da izazove DNK oštec'enje procenjena je primenom elektroforeze DNK pojedinačnih ćelija (SCGE) / komet testa. Ćelije pune krvi su bile izložene trima konačnim koncentracijama tiazofurina (1 µM/mL, 2 µM/mL, and 5 µM/mL) tokom 30 minuta in vitro. Rezultati. Naši rezultati ukazuju na to da je tiazofurin proizveo značajan nivo DNK oštec'enja na c'elijama pune krvi nakon 30 minuta izlaganja u odnosu na kontrolu. Sve ispitivane koncentracije su dovele do značajnog nastanka kometa, pri čemu je nivo oštećenja rastao s koncentracijom. Zaključak. Naše istraživanje c'elija tretiranih tiazofurinom i njihova reakcija na izazivanje DNK oštec'enja pokazalo je da je tiazofurin ispoljio genotoksični efekat pod opisanim uslovima

Assessment of adrenaline-induced DNA damage in whole blood cells with the comet assay

Harmful effects of elevated levels of catecholamines are mediated by various mechanisms, including gene transcription and formation of oxidation products. The aim of this study was to see whether the molecular mechanisms underlying the damaging action of adrenaline on DNA are mediated by reactive oxygen species (ROS). To do that, we exposed human whole blood cells to 10 mu mol L-1 adrenaline or 50 mu mol L-1 H2O2 (used as positive control) that were separately pre-treated or post-treated with 500 mu mol L-1 of quercetin, a scavenger of free radicals. Quercetin significantly reduced DNA damage in both pre- and post-treatment protocols, which suggests that adrenaline mainly acts via the production of ROS. This mechanism is also supported by gradual lowering of adrenaline and H2O2-induced DNA damage 15, 30, 45, and 60 min after treatment. Our results clearly show that DNA repair mechanisms are rather effective against ROS-mediated DNA damage induced by adrenaline

Evaluation of the antioxidant potential of Biochaga in vitro

Background: Antioxidants and prooxidants have an impact on the intracellular oxidative equilibrium. Overproduction of prooxidants leads to oxidative stress caused by imbalances in oxidative reduction pathways. The body can be supplied with non-enzymatic, low molecular weight antioxidants through diet.The edible medicinal mushroom Chaga, Inonotus obliquus (Ach. ex Pers.) Pilat, has long been long used to treat or prevent various health conditions and disorders. The bioactive compounds of Chaga exhibit antitumor, anti-inflammatory, hypoglycemic, immunomodulatory, antioxidant, and antigenotoxic effects. Material and Methods: DPPH (2,2-diphenyl-1-picrylhydrazyl) scavenging activity, FRAP (ferric reducing antioxidant power) total antioxidant activity, and hydroxyl radical scavenging capacity were measured. Results: Commercial Biochaga (B), a water extract of Biochaga mushroom, was obtained from Sibpribor Ooo, Irkutsk, Russia. B (IC =5.9 mg/ mL) showed moderate reducing power compared in comparison to vitamin C and strong compared to BTH. B (IC =1.78 mg/mL) showed remarkable free radical scavenging and moderate hydroxyl scavenging activity (IC =8.473 mg/mL). Conclusion: We can place Biochaga in the radical scavenging category because it efficiently eliminates hydroxyl radicals against which the body has insufficient antioxidant defenses.14th Balkan Congress of Human Genetics and 9 th Rare Disease SEE Meeting “Genetic Diseases from Diagnostics to Prevention and Therapy”(ABSTRACT BOOK), October 05-07, 2023, Hotel “DoubleTree by Hilton” Skopje, North Macedoni

Antigenotoxic effect of quercetin on thyroxine-induced DNA damage in human whole blood cells in vitro

The binding of thyroid hormones to specific nuclear receptors in target cells induces synthesis of enzymes associated with redox processes, leading to the formation of reactive oxygen species (ROS), which can cause damage of DNA molecule. Quercetin has already been shown to have protective effect against DNA damage, with its most pronounced feature being scavenging of free radicals. The aim of this study was to evaluate antigenotoxic potential of quercetin against thyroxine-induced DNA damage in human whole blood cells by using the comet assay. For that purpose, cells were exposed to 50 μM thyroxine and separately pre-treated or post treated with 500 μM of quercetin. Results showed that DNA damage was significantly reduced in cells pre-treated with this scavenger of free radicals. Obtained results indicate the ability of thyroxine to be a mediator of DNA damage and that quercetin displayed protective effect against thyroxine-induced genotoxicity.1st Congress of Geneticists in Bosnia and Herzegovina with International Participation Sarajevo, Bosnia and Herzegovina, 02nd-04th October, 2019, Book of abstract

Antigenotoxic and antioxidant potential of medicinal mushrooms (Immune Assist) against DNA damage induced by free radicals-an in vitro study

Immune Assist (IA) is produced from extract of six species of medical mushrooms: Agaricus blazei - Cordyceps sinensis - Grifola frondosa - Ganoderma lucidum - Coriolus versicolor - Lentinula edodes. The genoprotective potential of IA was evaluated for the first time. Significant antigenotoxic effects were detected in human peripheral blood cells against H2O2 induced DNA damage, in the pretreatment and in the posttreatment. The most efficient concentration of IA in pretreatment was 500 mu g/mL, while in posttreatment it was the concentration of 250 mu g/mL. Kinetics of attenuation of H2O2 induced DNA damage in posttreatment with the optimal concentration of IA showed significant decrease in the number of damaged cells at all time periods (15-60 min), reaching the greatest reduction after 15 and 45 min. Remarkable center dot OH scavenging properties and moderate reducing power, together with the modest DPPH scavenging activity, could be responsible for the great attenuation of DNA damage after 15 min of exposure to IA, while reduction of DNA damage after 45 min could be the result in additional stimulation of the cell's repair machinery. Our results suggest that IA displayed antigenotoxic and antioxidant properties. A broader investigation of its profile in biological systems is needed

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

Evaluation of antioxidative, antigenotoxic, and anticancer activities of commercial medical mushrooms products: Agaricus blazei, Cordyceps Sinensis and Immune Assist

Mushrooms have been evaluated for their nutritional and medicinal properties for centuries. We evaluated the biological activities of commercial products of Cordyceps Sinensis (CS), Agaricus blazei (AB), and Immune Assist (IA) (all produced by Aloha Medicinals). Immune Assist (IA) is made from extract of six species of medical mushrooms: Agaricus blazei - Cordycepssinensis - Grifola frondosa - Ganoderma lucidum - Coriolus versicolor - Lentinula edodes. The antioxidant evaluation showed that CS had strong OH scavenging properties and moderate reducing power, while its DPPH scavenging ability was weak. AB displayed remarkable ·OH scavenging properties, moderate reducing power, and modest DPPH scavenging activity, similar to IA. Also, CS, AB and IA displayed significant antigenotoxic effects in human peripheral blood cells against H2O2-induced DNA damage. Further, the cytotoxicity of CS, AB, and IA to HS-5 (bone marrow stroma cells), MCF- 7 (human breast epithelial cell line), and MDA-MB-231 (human breast carcinoma cell line) were assessed using MTT assay. AB and IA could inhibit the proliferative action of mentioned cancer cell lines after 72h in a dose-dependent manner, while there were no effects on HS-5 cell lines proliferation, while CS did not show anti-proliferative/ cytotoxic activity on cancer cell lines. Current findings remain a significant challenge for the usage of medicinal mushrooms in the field of cancer prevention/treatment, and thus an active area of future research.dodati broj ugovora za Institut Vinča51st European Environmental Mutagenesis and Genomics Society (EEMGS) & 27th Spanish Environmental Mutagenesis and Genomics Society (SEMA) meeting, May 15th- 18th, 202

Assessment of DNA damage in blood, liver and kidney cells in a hypertensive rat model using comet assay

Hypertension is one of the primary risk factors for heart disease and stroke, the leading causes of death worldwide. Numerous factors have been implicated in the pathophysiology of hypertension: endothelial dysfunction, arterial remodeling and vascular inflammation. Common to all these processes is increased bioavailability of reactive oxygen species in the vessels, heart, brain and kidneys. Oxidative stress and increased reactive oxygen species levels damage all macromolecules, with DNA being particularly susceptible to oxidative damage. The aim of this study was to determine whether there is a difference in the level of DNA damage between normotensive and hypertensive rats using the alkaline comet assay. Blood samples and cells suspension from liver and kidney from three male spontaneously hypertensive rats were obtained. Three normotensive male Wistar rats were used as a control. Increased level of DNA damage was detected in blood and both of the studied tissues of hypertensive rats compared to the control, where significant difference was present in the liver and kidney cell suspensions. These results indicate that untreated hypertension in rats leads to an increased DNA damage in all of the studied samples, detected by comet assay.1st Congress of Geneticists in Bosnia and Herzegovina with International Participation Sarajevo, Bosnia and Herzegovina, 02nd-04th October, 2019, Book of abstract