Andreja Leskovac

Toxic effects of diazinon and its photodegradation products

The toxic effects of diazinon and its irradiated solutions were investigated using cultivated human blood cells (lymphocytes and erythrocytes) and skin fibroblasts. Ultra Performance Liquid Chromatography (UPLC)-UV/VIS system was used to monitor the disappearance of starting diazinon during 115-min photodegradation and formation of its by-products (diazoxon and 2-isopropyl-6-methyl-4-pyrimidinol (IMP)) as a function of time. Dose-dependent AChE and Na(+)/K(+)-ATPase inhibition by diazinon was obtained for all investigated cells. Calculated IC(50) (72 h) values, in M, were: 7.5x10(-6)/3.4x10(-5), 8.7x10(-5)/6.6x10(-5), and 3.0x10(-5)/4.6x10(-5) for fibroblast, erythrocyte and lymphocyte AChE/Na(+)/K(+)-ATPase, respectively. Results obtained for reference commercially purified target enzymes indicate similar sensitivity of AChE towards diazinon (IC(50) (20 min)-7.8x10(-5)M), while diazinon concentrations below 10mM did not noticeably affect Na(+)/K(+)-ATPase activity. Besides, diazinon and IMP induced increasing incidence of micronuclei (via clastogenic mode of action) in a dose-dependent manner up to 2x10(-6)M and significant inhibition of cell proliferation and increased level of malondialdehyde at all investigated concentrations. Although after 15-min diazinon irradiation formed products do not affect purified commercial enzymes activities, inhibitory effect of irradiated solutions on cell enzymes increased as a function of time exposure to UV light and resulted in significant reduction of AChE (up to 28-45%) and Na(+)/K(+)-ATPase (up to 35-40%) at the end of irradiation period. Moreover, photodegradation treatment strengthened prooxidative properties of diazinon as well as its potency to induce cytogenetic damage.

Dysfunctional telomeres in primary cells from Fanconi anemia FANCD2 patients

BackgroundFanconi anemia (FA) is characterized by sensitivity to DNA cross-linking agents, mild cellular, and marked clinical radio sensitivity. In this study we investigated telomeric abnormalities of non-immortalized primary cells (lymphocytes and fibroblasts) derived from FA patients of the FA-D2 complementation group, which provides a more accurate physiological assessment than is possible with transformed cells or animal models.ResultsWe analyzed telomere length, telomere dysfunction-induced foci (TIFs), sister chromatid exchanges (SCE), telomere sister chromatid exchanges (T-SCE), apoptosis and expression of shelterin components TRF1 and TRF2. FANCD2 lymphocytes exhibited multiple types of telomeric abnormalities, including premature telomere shortening, increase in telomeric recombination and aberrant telomeric structures ranging from fragile to long-string extended telomeres. The baseline incidence of SCE in FANCD2 lymphocytes was reduced when compared to control, but in response to diepoxybutane (DEB) the 2-fold higher rate of SCE was observed. In contrast, control lymphocytes showed decreased SCE incidence in response to DEB treatment. FANCD2 fibroblasts revealed a high percentage of TIFs, decreased expression of TRF1 and invariable expression of TRF2. The percentage of TIFs inversely correlated with telomere length, emphasizing that telomere shortening is the major reason for the loss of telomere capping function. Upon irradiation, a significant decrease of TIFs was observed at all recovery times. Surprisingly, a considerable percentage of TIF positive cells disappeared at the same time when incidence of γ-H2AX foci was maximal. Both FANCD2 leucocytes and fibroblasts appeared to die spontaneously at higher rate than control. This trend was more evident upon irradiation; the percentage of leucocytes underwent apoptosis was 2.59- fold higher than that in control, while fibroblasts exhibited a 2- h delay before entering apoptosis.ConclusionThe results of our study showed that primary cells originating from FA-D2 patients display shorten telomeres, elevated incidence of T-SCEs and high frequency of TIFs. Disappearance of TIFs in early response to irradiation represent distinctive feature of FANCD2 cells that should be examined further.

Myeloperoxidase Inhibitors as Potential Drugs.

Myeloperoxidase (MPO) is an important member of the haem peroxidase - cyclooxygenase superfamily. This enzyme is physiologically expressed in circulating neutrophils, monocytes and some tissue macrophages including microglia. MPO plays an essential role in the antimicrobial and antiviral system of humans. The microbicidal activity of MPO exists due to its capability to oxidize halide and pseudohalide ions (CI(-), Br(-), I(-) and SCN(-)) by H2O2, thereby producing respective hypohalous acids (HOX). During the phagocytosis of pathogens, azurophilic granules release their content together with MPO into phagolysosomes. On the other hand, MPO can be discharged outside the phagocytes. Due to this, tissue damage during inflammation is greatly promoted by MPO-derived oxidants. Regarding its activity, MPO is a key factor in a great number of conditions within the group of cardiovascular diseases, inflammatory diseases, neurodegenerative diseases, kidney diseases and immune-mediated diseases. Therefore, MPO and its downstream inflammatory pathways might be attractive targets for both prognostic and therapeutic intervention in the prophylaxis of all mentioned illnesses. Nowadays, structure and reaction mechanism of MPO are known, which enable rational strategy in the development of specific MPO inhibitors that still preserve MPO activity during host defense from bacteria, but hinder pathophysiologically persistent activation of MPO. Various methods for MPO activity inhibition and unfavorable effects of MPO-derived oxidants remodeling will be discussed. Emphasis will be put on various known inhibitors, as well as on newly investigated natural products, which can also inhibit MPO activity.

Standing at the Gateway to Europe - The Genetic Structure of Western Balkan Populations Based on Autosomal and Haploid Markers

Contemporary inhabitants of the Balkan Peninsula belong to several ethnic groups of diverse cultural background. In this study, three ethnic groups from Bosnia and Herzegovina - Bosniacs, Bosnian Croats and Bosnian Serbs - as well as the populations of Serbians, Croatians, Macedonians from the former Yugoslav Republic of Macedonia, Montenegrins and Kosovars have been characterized for the genetic variation of 660 000 genome-wide autosomal single nucleotide polymorphisms and for haploid markers. New autosomal data of the 70 individuals together with previously published data of 20 individuals from the populations of the Western Balkan region in a context of 695 samples of global range have been analysed. Comparison of the variation data of autosomal and haploid lineages of the studied Western Balkan populations reveals a concordance of the data in both sets and the genetic uniformity of the studied populations, especially of Western South-Slavic speakers. The genetic variation of Western Balkan populations reveals the continuity between the Middle East and Europe via the Balkan region and supports the scenario that one of the major routes of ancient gene flows and admixture went through the Balkan Peninsula.

Radioprotective Effects of Gentianella austriaca Fractions and Polyphenolic Constituents in Human Lymphocytes

The aim of this study was to identify active principles of Gentianella austriaca responsible for the reduction of the incidence of micronuclei in irradiated lymphocytes in vitro. The radioprotective effects of ether (EF) and methanolic (MeF) fractions, water-soluble xanthones demethylbellidifolin (1), demethylbellidifolin 8-O-glucoside (2), bellidifolin 8-O-glucoside (3), and flavonoid swertisin (4) against chromosomal damage induced by gamma-rays were determined using the micronucleus test. EF and MeF showed better protection in treatment of human lymphocytes after gamma-irradiation than did isolated compounds. Among the isolated compounds, the effectiveness in reduction of the frequency of micronuclei followed the order 4>3>2>1. The anti-lipoperoxidant activity was in the order 2>4>1, while 3 slightly increased the level of malondialdehyde. In addition, the effectiveness in induction of apoptosis followed the order, 3>2>4, while 1 had no proapoptotic effect. These results suggest that the antioxidative properties of the polyphenols tested may contribute to the radioprotective effects of G. austriaca.

Gender-related differences in the oxidant state of cells in Fanconi anemia heterozygotes

Abstract Fanconi anemia (FA) is a rare cancer-prone genetic disorder characterized by progressive bone marrow failure, chromosomal instability and redox abnormalities. There is much biochemical and genetic data, which strongly suggest that FA cells experience increased oxidative stress. The present study was designed to elucidate if differences in oxidant state exist between control, idiopathic bone marrow failure (idBMF) and FA cells, and to analyze oxidant state of cells in FA heterozygous carriers as well. The results of the present study confirm an in vivo prooxidant state of FA cells and clearly indicate that FA patients can be distinguished from idBMF patients based on the oxidant state of cells. Female carriers of FA mutation also exhibited hallmarks of an in vivo prooxidant state behaving in a similar manner as FA patients. On the other hand, the oxidant state of cells in FA male carriers and idBMF families failed to show any significant difference vs. controls. We demonstrate that the altered oxidant state influences susceptibility of cells to apoptosis in both FA patients and female carriers. The results highlight the need for further research of the possible role of mitochondrial inheritance in the pathogenesis of FA.

Biological effects of Echinacea purpurea on human blood cells.

Biological Effects of Echinacea Purpurea on Human Blood Cells The aim of this study was to investigate radioprotective properties of Echinacea purpurea tablets in vivo. We analysed lymphocyte chromosome aberrations (CA), micronuclei (MN), apoptosis of leukocytes and haematological parameters in a group of radiation workers who were identified as carrying dicentric chromosomes in their lymphocytes. All radiation workers were taking two 275 mg Echinacea tablets b.i.d., according to a pharmacists recommendation. All parameters were analysed before and after the two-week treatment. At the end of the treatment lymphocyte CA frequency dropped significantly, and the number of apoptotic cells increased. The inverse lymphocyte-to-granulocyte ratio at the beginning of the study changed to normal at its end. In conclusion, biological effects observed after administration of Echinacea purpurea preparation suggest that it may be beneficial for the prevention of adverse health effects in workers exposed to ionising radiation. Biološki učinciI Echinacea purpurea na krvne stanice ljudi

Modulators of Acetylcholinesterase Activity: from Alzheimer's Disease to Anti-cancer Drugs.

BACKGROUND Acetylcholinesterase (AChE) is involved in the termination of impulse transmission by rapid hydrolysis of the neurotransmitter acetylcholine in numerous cholinergic pathways in the central and peripheral nervous systems. The enzyme inactivation leads to acetylcholine accumulation, hyperstimulation of nicotinic and muscarinic receptors, and disrupted neurotransmission. Hence, acetylcholinesterase inhibitors, interacting with the enzyme as their primary target, are applied as relevant drugs for different neurodegenerative diseases (such as Alzheimers and Parkinsons) as well as toxins. At the same time, there are increasing evidence that in non-neuronal context, AChE is involved in the regulation of cell proliferation, differentiation, apoptosis and cell-cell interaction. An irregular expression of AChE has been found in different types of tumors, suggesting the involvement of AChE in the regulation of tumor development. Having all this in mind, there is a possibility that some AChE inhibitors could be used as anti-cancer agents. OBJECTIVE This contribution will discuss a broad range of possible application of different AChE inhibitors as drugs, from well-known anti-Alzheimers disease drugs to their use in cancer treatment in future. Emphasis will be put on various known AChE inhibitors classes, whose application as drugs could be controversy, as well as on newly investigated natural products, which can also modulate AChE activity. CONCLUSION It is not clear a patient treated for neurodegenerative condition prone to increased risk for some types of cancer and vice versa. This is necessary to keep in mind during rational drug design process for all therapies, which are based on AChE as a target molecule.

Radiation-induced mitotic catastrophe in FANCD2 primary fibroblasts

Abstract Purpose: As the Fanconi anemia (FA) pathway is required for appropriate cell cycle progression through mitosis and the completion of cell division, the aim of the present study was to determine the destiny of FA cells after irradiation in vitro and to elucidate any difference in radiosensitivity between FA and control cells. Materials and methods: Analyses of phosphorylated histone H2AX (γ-H2AX) foci, micronuclei formation and cell cycle analysis were performed in unirradiated (0 min) and irradiated primary FA fibroblasts and in a control group at different post-irradiation times (30 min, 2 h, 5 h and 24 h). Results: The accumulation of γ-H2AX foci in irradiated FA fibroblasts was observed. At 24 h post-irradiation, 57% of FA cells were γ-H2AX foci-positive, significantly higher than in the control (p < 0.01). The cell cycle analysis has shown the transient G2/M arrest in irradiated FA fibroblasts. The portion of cells in the G2/M phase showed initial increase at 30 min post-irradiation and afterwards decreased over time reaching the pretreatment level 24 h after irradiation. Irradiated FA fibroblasts progressed to abnormal mitosis, as is shown by the production of cells with different nuclear morphologies from binucleated to multinucleated surrounded with micronuclei, and also by a high percentage of foci-positive micronuclei. The majority of radiation-induced micronuclei were γ-H2AX foci-positive, indicating that radiation-induced micronuclei contain fragments of damaged chromosomes. In contrast, in the control group, most of the micronuclei were classified as γ-H2AX foci-negative, which indicates that cells with unrepaired damage were blocked before entering mitosis. Conclusion: The results clearly indicate that mitotic catastrophe might be an important cell-death mechanism involved in the response of FA fibroblasts to ionizing radiation.

The impact of concentration and administration time on the radiomodulating properties of undecylprodigiosin in vitro

Abstract Undecylprodigiosin pigment (UPP) is reported to display cytotoxic activity towards various types of tumours. Nevertheless, its efficacy in modifying the cellular response to ionising radiation is still unknown. In this study, the radiomodulating effects of UPP were investigated. The effects of UPP were assessed in vitro by treating cultures of human peripheral blood with UPP and ionising radiation using two treatment regimens, the UPP pre-irradiation treatment and UPP post-irradiation treatment. The activity of UPP was investigated evaluating its effects on the radiation-induced micronuclei formation, cell proliferation, and induction of apoptosis. The redox modulating effects of UPP were examined measuring the catalase activity and the level of malondialdehyde, as a measure of oxidative stress. The results showed that UPP effects on cellular response to ionising radiation depend on its concentration and the timing of its administration. At low concentration, the UPP displayed radioprotective effects in γ-irradiated human lymphocytes while at higher concentrations, it acted as a radiosensitiser enhancing either mitotic catastrophe or apoptosis depending on the treatment regimen. The UPP modified redox processes in cells, particularly when it was employed prior to γ-irradiation. Our data highlight the importance of further research of the potential of UPP to sensitize tumour cells to radiation therapy by inhibiting pathways that lead to treatment resistance.