Gordana Joksić

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.

Inhibition of myeloperoxidase and antioxidative activity of Gentiana lutea extracts

The aim of this study was to investigate the inhibitory activity of Gentiana lutea extracts on the enzyme myeloperoxidase (MPO), as well as the antioxidant activity of these extracts and their correlation with the total polyphenol content. Extracts were prepared using methanol (100%), water and ethanol aqueous solutions (96, 75, 50 and 25%v/v) as solvents for extraction. Also, isovitexin, amarogentin and gentiopicroside, pharmacologically active constituents of G. lutea were tested as potential inhibitors of MPO. Antioxidant activity of extracts was determined using the 2,2-diphenyl-1-picrylhydrazyl (DPPH) scavenging test and also using cyclic voltammetry (CV). Among all extracts, the antioxidant capacity of 50% ethanol aqueous extract was the highest, both when measured using the DPPH test, with IC(50)=20.6 μg/ml, and when using CV. Also, 50% ethanol extract, showed the best inhibition of MPO activity in comparison with other extracts. In the group of the selected G. lutea constituents, gentiopicroside has proved to be the strongest inhibitor of MPO, with IC(50)=0.8 μg/ml. Also, the concentration of G. lutea constituents were determined in all extracts, using Ultra Performance Liquid Chromatography (UPLC).

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.

Randomized, placebo-controlled, double-blind trial of Swedish snus for smoking reduction and cessation

BackgroundEpidemiological studies suggest that smokeless tobacco in the form of Swedish snus has been used by many smokers in Scandinavia to quit smoking, but the efficacy of snus has so far not been evaluated in controlled clinical trials.MethodsWe conducted a randomized, double-blind, placebo-controlled, clinical trial aimed at assessing the efficacy of snus to help adult cigarette smokers in Serbia to substantially reduce, and, eventually, completely stop smoking. The study enrolled 319 healthy smokers aged 20-65 years at two occupational health centers in Belgrade, Serbia. Most of them (81%) expressed an interest to quit rather than just reduce their smoking. Study products were used ad libitum throughout the 48-week study period. The main study objective during the first 24 weeks was smoking reduction. The primary end-point was defined as a biologically verified reduction of ≥ 50% in the average number of smoked cigarettes per day during week 21-24 compared to baseline. During week 25-48 participants were actively instructed to stop smoking completely. Outcome measures of biologically verified, complete smoking cessation included 1-week point prevalence rates at clinical visits after 12, 24, 36, and 48 weeks, as well as 4-, 12- and 24-week continued cessation rates at the week 36 and 48 visits.ResultsAt the week 24 visit, the proportion of participants who achieved the protocol definition of a ≥ 50% smoking reduction was similar in the two treatment groups. However, the proportion that reported more extreme reductions (≥ 75%) was statistically significantly higher in the snus group than in the placebo group (p < 0.01). The results for biologically verified complete cessation suggested that participants in the snus group were more likely to quit smoking completely than the controls; the odds ratio (snus versus placebo) for the protocol estimates of cessation varied between 1.9 to 3.4, but these ratios were of borderline significance with p-values ranging from 0.04-0.10. Snus was well tolerated and only 2/158 (1.3%) participants in the snus group discontinued treatment due to an adverse event (in both cases unrelated to snus).ConclusionsSwedish snus could promote smoking cessation among smokers in Serbia, that is, in a cultural setting without traditional use of oral, smokeless tobacco.Trial registrationwww.clinicaltrials.gov, identifier: NCT00601042

Effects of Digoxin and Gitoxin on the Enzymatic Activity and Kinetic Parameters of Na+/K+-ATPase

Inhibition of Na+/K+-ATPase activity from human erythrocyte membranes and commercial porcine cerebral cortex by in vitro single and simultaneous exposure to digoxin and gitoxin was investigated to elucidate the difference in the mechanism of the enzyme inhibition by structurally different cardiac glycosides. The drugs exerted a biphasic dose-dependent inhibitory effect on the enzyme activity in both tissues, supporting the existence of two sensitive Na+/K+-ATPase isoforms. The IC50 values for the low and high affinity isoforms were calculated from the inhibition curves using mathematical analysis. The Hill coefficient (n) fulfilled the relationship 1<n<3, suggesting cooperative binding of inhibitors to the enzyme. Kinetic analysis showed that digoxin and gitoxin inhibited Na+/K+-ATPase by reducing the maximum enzymatic velocity (Vmax) and Km, implying an uncompetitive mode of interaction. Both the isoforms were always more sensitive to gitoxin. The erythrocyte enzyme was more sensitive to the inhibitors in the range of low concentrations but the commercial cerebral cortex enzyme exerted a higher sensitivity in high inhibitors affinity concentration range. By simultaneous exposure of the enzyme to digoxin and gitoxin in combinations a synergistic effect was achieved by low inhibitor concentrations. An antagonistic effect was obtained with erythrocyte membrane enzyme at high inhibitors concentration.

Plasma induced DNA damage: Comparison with the effects of ionizing radiation

We use human primary fibroblasts for comparing plasma and gamma rays induced DNA damage. In both cases, DNA strand breaks occur, but of fundamentally different nature. Unlike gamma exposure, contact with plasma predominantly leads to single strand breaks and base-damages, while double strand breaks are mainly consequence of the cell repair mechanisms. Different cell signaling mechanisms are detected confirming this (ataxia telangiectasia mutated - ATM and ataxia telangiectasia and Rad3 related - ATR, respectively). The effective plasma doses can be tuned to match the typical therapeutic doses of 2 Gy. Tailoring the effective dose through plasma power and duration of the treatment enables safety precautions mainly by inducing apoptosis and consequently reduced frequency of micronuclei.

Inhibition of Aldose Reductase by Gentiana lutea Extracts

Accumulation of intracellular sorbitol due to increased aldose reductase (ALR2) activity has been implicated in the development of various secondary complications of diabetes. Thus, ALR2 inhibition could be an effective strategy in the prevention or delay of certain diabetic complications. Gentiana lutea grows naturally in the central and southern areas of Europe. Its roots are commonly consumed as a beverage in some European countries and are also known to have medicinal properties. The water, ethanol, methanol, and ether extracts of the roots of G. lutea were subjected to in vitro bioassay to evaluate their inhibitory activity on the ALR2. While the ether and methanol extracts showed greater inhibitory activities against both rat lens and human ALR2, the water and ethanol extracts showed moderate inhibitory activities. Moreover, the ether and methanol extracts of G. lutea roots significantly and dose-dependently inhibited sorbitol accumulation in human erythrocytes under high glucose conditions. Molecular docking studies with the constituents commonly present in the roots of G. lutea indicate that a secoiridoid glycoside, amarogentin, may be a potential inhibitor of ALR2. This is the first paper that shows G. lutea extracts exhibit inhibitory activity towards ALR2 and these results suggest that Gentiana or its constituents might be useful to prevent or treat diabetic complications.

Inhibition of Vascular Smooth Muscle Cell Proliferation by Gentiana lutea Root Extracts

Gentiana lutea belonging to the Gentianaceae family of flowering plants are routinely used in traditional Serbian medicine for their beneficial gastro-intestinal and anti-inflammatory properties. The aim of the study was to determine whether aqueous root extracts of Gentiana lutea consisting of gentiopicroside, gentisin, bellidifolin-8-O-glucoside, demethylbellidifolin-8-O-glucoside, isovitexin, swertiamarin and amarogentin prevents proliferation of aortic smooth muscle cells in response to PDGF-BB. Cell proliferation and cell cycle analysis were performed based on alamar blue assay and propidium iodide labeling respectively. In primary cultures of rat aortic smooth muscle cells (RASMCs), PDGF-BB (20 ng/ml) induced a two-fold increase in cell proliferation which was significantly blocked by the root extract (1 mg/ml). The root extract also prevented the S-phase entry of synchronized cells in response to PDGF. Furthermore, PDGF-BB induced ERK1/2 activation and consequent increase in cellular nitric oxide (NO) levels were also blocked by the extract. These effects of extract were due to blockade of PDGF-BB induced expression of iNOS, cyclin D1 and proliferating cell nuclear antigen (PCNA). Docking analysis of the extract components on MEK1, the upstream ERK1/2 activating kinase using AutoDock4, indicated a likely binding of isovitexin to the inhibitor binding site of MEK1. Experiments performed with purified isovitexin demonstrated that it successfully blocks PDGF-induced ERK1/2 activation and proliferation of RASMCs in cell culture. Thus, Gentiana lutea can provide novel candidates for prevention and treatment of atherosclerosis.

Pallister-Killian Syndrome: Rare Phenotypic Features and Variable Karyotypes

Pallister-Killian Syndrome: Rare Phenotypic Features and Variable Karyotypes

Plasma levels of insulin-like growth factor-I, insulin-like growth factor binding protein-1, coenzyme Q10 and vitamin E in female populations from Poland, Serbia and Sweden

Exposure to environmental contaminants such as polycyclic aromatic hydrocarbons (PAHs), life style and nutritional status of a population are important factors that may influence normal serum levels of antioxidants and the insulin-like growth factor system. In this study we examined serum levels of insulin-like growth factor-I (IGF-I), insulin-like growth factor binding protein-1(IGFBP-1), coenzyme Q10 (CoQ) and vitamin E in healthy female populations (n=4 x 100) aged 19-59 years from Poland (PL), Sweden (SE), Serbia I (SR I) and Serbia II (SR II). The last group lived in an environmental emergency area affected by the bombings of 1999 in Serbia. The Polish and SR I cohorts exhibited low IGFSD-score levels, (-2 to +/-0), compared to females from SE with IGFSD-score 0. In the SR II population, the IGFSD range was between -1 and 1. The IGFBP-1 levels of the Polish and SR I groups were lower than in the Swedish population, while the SR II levels showed a broader distribution, 20-80 microg/l. The CoQ values in the Swedish and Polish samples were around 1 nmol/ml. In contrast, the SR I cohorts exhibited higher concentrations, 1.5-3.5 nmol/ml and the SR II group had extremely low levels, <0.5 nmol/ml. The vitamin E concentrations were similar in the Polish and Swedish populations, 20-40 nmol/ml, while it was twice as high, 40-80 nmol/ml in the SR I and very low in the SR II group, which is half of the Polish and Swedish cohorts. These results suggest that different lifestyles and environmental factors affect both the IGF system and the antioxidants CoQ10 and vitamin E in female populations in Europe. The females living in the polluted area had different patterns of both the IGF and antioxidant systems. These findings may explain differences in morbidity and mortality in these countries.