Cytogenetic alterations in peripheral cells of Alzheimer’s disease patients

Alzheimer’s disease (AD) is the most frequent progressive neurodegenerative disorder in elderly associated with irreversible cognitive impairment and dementia. The vast majority of AD patients are sporadic (SAD) in which the disease develops after age of 65. Despite of century of research, we lack understanding of the SAD etiology and pathogenesis. Several hypotheses try to explain the main causes of brain degeneration in SAD, one of them assuming that genomic instability and the reentry of certain neurons into the incomplete cell cycle may be the pathogenic basis of the disease. Although the brain is the most affected organ in AD, numerous studies showed structural and functional alterations in peripheral tissues, suggesting that AD is a generalized systemic disorder. Diverse changes in peripheral cells from AD patients are described in literature including cell cycle aberration and chromosome instability, alterations in cell viability, proliferation and apoptosis, oxidative metabolism, amyloid precursor protein and amyloid β protein metabolism, and other cellular processes. The aim of this paper was to summarize and review the results of our investigations and the growing literature data concerning the multiple chromosomal alterations in peripheral cells of AD patients and to consider their possible role in the disease pathogenesis as well as the importance of such investigations. [Projekat Ministarstva nauke Republike Srbije, br. 173034

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

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

Homeostaza gvožđa

In this paper we described the homeostasis of elementary iron: its absorption, utilization, storage and elimination. Special attention was paid to new insights into the regulatory mechanisams involved in its homestasis, especially the role of recently discovered hormone hepcidin, which is synthesized in the liver. Since it is reckoned that hepcidin has a central role in the systemic regulation of iron metabolism, the influences of various factors and conditions on the hepatic synthesis of hepcidin were delineated. Furthermore, we briefly described the most common disorders of iron homeostasis and their possible involvement in the pathogenesis of some diseases.U radu je opisan promet gvožđa u organizmu: unošenje, iskorišćavanje, deponovanje i eliminacija. Posebna pažnja posvećena je novim saznanjima o regulaciji prometa tog elementa neophodnog za život. U tom smislu je istaknut, relativno skoro otkriven, hormon hepcidin koji sintetišu hepatociti. Smatra se da hepcidin zauzima centralno mesto u sistemskoj regulaciji metabolizma gvožđa i prikazan je uticaj različitih faktora i stanja na sintezu hepcidina u jetri. Ukratko su opisani i najčešći poremećaji homeostaze gvožđa i njegova eventualna uloga u patogenezi nekih oboljenja

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

Kinetika proliferacije i citogenetičke promene u humanim limfocitima pod dejstvom estradiola in vitro

Metabolic conversion of oestrogen phenolic groups may create conditions of oxidative stress accompanied by damage of cellular macromolecules including DNA. The aim of this investigation was to evaluate the cell cycle kinetics and possible cytogenetic changes in cultured human peripheral blood lymphocytes exposed to seven experimental concentrations of 17β-oestradiol (range 10-10 M to 10-4 M). Cell cycle kinetics was analyzed on metaphase spreads prepared for a standard analysis of sister-chromatid exchanges (SCEs) stained by fluorescent-plus-Giemsa (FPG) technique. Cytogenetic changes were monitored by analysis of chromosome damage (gaps and breaks), structural and numerical aberrations. On the basis of the obtained results it can be concluded that oestradiol has no significant influence on cell cycle kinetics and mitotic index of cultured human lymphocytes. However, at estradiol concentration of 7×10-6 M, and at higher concentrations used in this experiment, there was a significant increase of gaps, breaks and aneuploidies. On the other hand, oestradiol treatment has not changed the frequency of polyploid cells. Therefore, it can be concluded that high concentrations of oestradiol pose some genetic risk detectable at cytogenetic level.Metabolička konverzija fenolnih grupa estrogenih hormona može da dovede do oksidativnog stresa praćenog oštećenjima različitih makromolekula u eliji, uključujući DNK. Cilj ovog istraživanja je evaluacija kinetike proliferacije i mogućih citogenetičkih promena u kulturama humanih limfocita pod dejstvom sedam eksperimentalnih koncentracija 17β-estradiola (opseg od 10-10M do10-4 M). Kinetika proliferacije limfocita analizirana je na metafaznim figurama obojenim tehnikom FPG za standardne analize razmena sestrinskih hromatida (SCE). Citogenetičke promene praćene su analizama hromozomskih oštećenja (gapovi i prekidi), strukturnih i numeričkih aberacija hromozoma. Na osnovu dobijenih rezultata može se zaključiti da estradiol ne utiče značajno na mitotsku aktivnost i kinetiku proliferacije limfocita u kulturi. Međutim, pri koncentraciji od 7×10-6 M, kao i pri višim eksperimentalnim koncentracijama korišćenim u ovim eksperimentima, zapažen je porast gapova, prekida i aneuploidija. S druge strane, tretman estradiolom ne menja učestalost poliploidnih ćelija. Prema tome, može se zaključiti da visoke koncentracije estradiola izazivaju izvestan genetički rizik koji se može detektovati na citogenetičkom nivou

Examination of the effects of X-ray phase contrast imaging dose on DNA in mesenchymal stem cells by comet assay

INTRODUCTION: Imaging techniques based on X-ray phase-contrast (XPC) have shown tremendous promise for applications involving biomaterials and soft tissue formation [1,2].XPC imaging can be applied at higher energy offering the potential for lower dose imaging. Essential to the development of this technique and its routine use is an understanding of the potential damage of X-ray dose on cells and tissues. EXPERIMENTAL: In this study the comet assay, a sensitive assay for DNA damage, was used to evaluate DNA damage on mesenchymal stem cells (MSCs) exposedto X-ray irradiation. We examined the effects of early (immediately following irradiation) and delayed (24h post-irradiation) X-ray effects caused by low (15mGy) and intermediate (150mGy and 1.5 Gy) exposure on MSCs during a monitoring period of 4 weeks (five irradiations, one weekly). Cells were submitted to apolychromatic X-ray source (Thermo Fisher PXS10 conditions: voltage 45 kV, source current 160A, source power 7.2 W, source spot size 9 um, photon flux on the sample 7.66106photonss-1mm-2irradiation).Statistical analysis was performedby using Two-way analysis of variance (ANOVA) with Tukey’s multiple comparisons posttest in GraphPad Prism 5.0.A difference at p< 0.05 was considered statistically significant.RESULTS AND DISCUSSION: Resultsof the DNA comet assay indicated that early effects of low-and intermediate-dose of XPC induced an increase in the number of cells with DNA damage after each irradiation, where intermediate-dose (150 mGy and 1.5 Gy) produced significantly higher damage relative to controls. DNA damage induced by low and intermediate doses returned to the control value 24h after the irradiation exposure, suggesting a strong protection of MSCs at the tested doses of XPC irradiation. CONCLUSIONS: The data presented in this studyshows that 24 h after the last of five weekly low and intermediate doses XPC irradiation, the harmful effects on DNA in MSCs were notdetected. The current study reinforces the need of investigating consequences of low and intermediate doses of X-ray PC irradiation in the field of tissue engineering and provide new basis for MSCs using in the clinics.ExcellMater Conference 2024: Innovative Biomaterials for Novel Medical Devices, Belgrade, Serbia, April 10-12, 2024

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

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