Evaluation of novel dendrimer-gold complex nanoparticles for theranostic application in oncology.

Aim: Despite some successful examples of therapeutic nanoparticles reaching clinical stages, there is still a significant need for novel formulations in order to improve the selectivity and efficacy of cancer treatment. Methods: The authors developed two novel dendrimer-gold (Au) complex-based nanoparticles using two different synthesis routes: complexation method (formulation A) and precipitation method (formulation B). Using a biomimetic cancer-on-a-chip model, the authors evaluated the possible cytotoxicity and internalization by colorectal cancer cells of dendrimer-Au complex-based nanoparticles. Results: The results showed promising capabilities of these nanoparticles for selectively targeting cancer cells and delivering drugs, particularly for the formulation A nanoparticles. Conclusion: This work highlights the potential of dendrimer-Au complex-based nanoparticles as a new strategy to improve the targeting of anticancer drugs.

Synthesis, characterization, antitumor potential, and investigation of mechanism of action of copper(ii) complexes with acylpyruvates as ligands: interactions with biomolecules and kinetic study.

Considering the urgency of finding a cure for vicious diseases such as tumors, we have synthesized and characterized a small series of new copper(ii) complexes with biologically important ligands such as acylpyruvate. In addition to this, we used another four copper(ii) complexes, with ligands of the same type to examine the antitumor potential. The antitumor potential of the copper(ii) complexes was examined on three tumor cell lines and one normal human cell line using the MTT assay. All seven tested complexes showed very good cytotoxic effects. Two copper complexes that showed the best antitumor potential were selected for further testing that showed the best potential for potential application in the future. The mechanism of activity of these complexes was examined in detail using tests such as cell cycle, ROS level, oxidative DNA damage, and proteins related to hypoxia analysis. In addition, we examined the binding abilities of these complexes with biomolecules (Guo, Ino, 5'-GMP, BSA, and DNA). The results showed that the tested compounds bind strongly to DNA molecules through intercalation. Also, it has been shown that the tested compounds adequately bind to the BSA molecule, which indicates an even greater potential for some future application of these compounds in clinical practice.

Gold(III) Complexes with Phenanthroline-derivatives Ligands Induce Apoptosis in Human Colorectal and Breast Cancer Cell Lines.

Due to their promising effects, gold(III) complexes recently drew increasing attention in the design of new metal-based anticancer therapeutics. Two gold(III) complexes, square-planar [Au(DPP)Cl2]+ - Complex 1 and distorted square-pyramidal [Au(DMP)Cl3] - Complex 2 (where DPP=4,7-diphenyl-1,10-phenanthroline and DMP=2,9-dimethyl-1,10-phenanthroline) were previously synthetized, described and approved as complexes with pronounced cytotoxic effects on colorectal HCT-116 and breast MDA-MB-231 cancer cells. This study investigated the type of cell death by AO/EB double staining, and identification of possible targets responsible for their cytotoxicity, monitored by immunofluorescence and qPCR methods. Both complexes induced apoptosis in all applied concentrations. In the HCT-116 cells apoptosis was activated by external apoptotic pathway, via increase of Fas receptor protein expression and Caspase 8 gene expression. Also, the mitochondrial pathway was triggered by affecting the Bcl-2 members of regulatory proteins and increased caspase 9 protein expression. In MDA-MB-231 cells, apoptosis was initiated from the mitochondria, due to disbalance between expressions of pro- and anti-apoptotic Bcl-2 family members and caspase 9 activation. Complex 1 shows better activity compared to Complex 2, which is in accordance with its structural characteristics. The results deal weighty data about proapoptotic activity of gold(III) complexes and highlighted potential targets for cancer therapy.

Synthesis, characterization, DNA interactions and biological activity of new palladium(II) complexes with some derivatives of 2-aminothiazoles.

Newly palladium(II) complexes (C1, C2) with derivatives of 2-aminothiazoles (L1 = 2-amino-6-methylbenzothiazole, L2 = 2-amino-6-chlorobenzothiazole), general formula [PdL2Cl2] were synthesized and characterized by elemental microanalyses, IR, NMR spectroscopy and X-ray spectroscopy in case of [Pd(L2)2Cl2]. The kinetic of the substitution reactions of complexes and the nucleophiles, such as guanosine-5'-monophosphate (5'-GMP), tripeptide glutathione (GSH) and amino acid L-methionine (L-Met), were studied by stopped-flow technique. The complex C2 was always more reactive, while the order of the reactivity of the nucleophiles, due to the associative mode of the reaction, was L-Met > GSH > 5'-GMP. In order to determine the type of interactions between palladium(II) complexes and calf thymus DNA (CT-DNA), we used electronic absorption spectroscopy, viscosity measurements, and fluorescence spectroscopic studies, while interactions with bovine serum albumin (BSA) were determined only with fluorescence spectroscopic studies. The observed results confirmed that both complexes bound to DNA by groove binding. The significantly strong interaction with BSA, especially for complex C2, was also observed. In vitro cytotoxic activity was evaluated against four tumor cell lines, 4 T1, CT26, MDA-MB-468, HCT116 and mesenchymal stem cells (mMSC). C1 complex showed higher cytotoxic activity against CT26 cell line. Flow cytometry analysis showed that C1 stimulated apoptosis of tumor cells via inhibition of expression of antiapoptotic Bcl-2 molecule and decelerated proliferation by decreasing Cyclin-D and increasing expression of P21. In vitro antimicrobial activity for ligands and corresponding palladium(II) complexes was investigated by microdilution method and minimum inhibitory concentration (MIC) and minimum microbicidal concentration (MMC) were determined. Tested compounds exhibited selective and moderate activity.

Bis(triazinyl)pyridine complexes of Pt(II) and Pd(II): studies of the nucleophilic substitution reactions, DNA/HSA interactions, molecular docking and biological activity.

Four new complexes of Pt(II) and Pd(II), [Pd(L1)Cl]Cl 1, [Pd(L2)Cl]Cl 2, [Pt(L1)Cl]Cl 3 and [Pt(L2)Cl]Cl 4 (where L1 = 2,6-bis(5,6-diphenyl-1,2,4-triazin-3-yl)pyridine and L2 = 2,6-bis(5,6-dipropyl-1,2,4-triazin-3-yl)pyridine), were synthesized. Characterization of the complexes was performed using elemental analysis, IR, 1H NMR spectroscopy and MALDI-TOF mass spectrometry. The substitution reactions of 1-4 complexes with L-methionine (L-met), L-cysteine (L-cys) and guanosine-5'-monophosphate (5'-GMP), were studied spectrophotometrically at physiological conditions. Complexes with ligand L1 (1 or 3) were more reactive than those with ligand L2 (2 or 4) by a factor ranging up to 1.57 and 3.71, respectively. The order of reactivity of the nucleophiles was: L-met > L-cys > 5'-GMP. The interactions of complexes with calf thymus-DNA (CT-DNA) and human serum albumin (HSA) were studied by Uv-Vis absorption and fluorescence emission spectroscopy. Competitive binding studies with intercalative agent ethidium bromide (EB) and minor groove binder Hoechst 33258 were performed as well. All studied complexes can interact with DNA through the intercalation and minor groove binding, where the latter was preferred. The binding constants (103 and 104 M-1) for the interaction of complexes with HSA indicate the moderate binding affinity of complexes 1-4 to protein. The trends in the experimental results of binding studies between complexes 3 and 4 with DNA and HSA were compared to those obtained from the molecular docking study. Biological evaluation of cytotoxicity of 1 and 2 on HCT-116 and MDA-MB-231 cell lines showed significant cytotoxic and prooxidative character, while 2 also exerted extraordinary selectivity towards colon cancer in comparison to breast cancer cells. The nucleophilic substitution reactions, DNA/HSA interactions, molecular docking and biological activity of bis(triazinyl)pyridine complexes of Pt(II) and Pd(II) were studied.

Gold(III) Complexes: An Overview on Their Kinetics, Interactions With DNA/BSA, Cytotoxic Activity, and Computational Calculations.

In the last few years, metallodrugs play a key role in the development of medicinal chemistry. The choice of metal ion, its oxidation state and stability, and the choice of inert and labile ligands are just some of the very important facts which must be considered before starting the synthesis of complexes with utilization in medicinal purpose. As a result, a lot of compounds of different transition metal ions found application for diagnostic and therapeutic purpose. Beside all, gold compounds have attracted particular attention. It is well-known that gold compounds could be used for the treatment of cancer, HIV, rheumatoid arthritis (chrysotherapy), and other diseases. This metal ion has unoccupied d-sublevels and possibility to form compounds with different oxidation states, from -1 to +5. However, gold(I) and gold(III) complexes are dominant in chemistry and medicine. Especially, gold(III) complexes are of great interest due to their structural similarity with cisplatin. Accordingly, this review summarizes the chemistry of some mononuclear and polynuclear gold(III) complexes. Special attention is given to gold(III) complexes with nitrogen-donor inert ligands (aliphatic or aromatic that have a possibility to stabilize complex) and their kinetic behavior toward different biologically relevant nucleophiles, mechanism of interaction with DNA/bovine serum albumin (BSA), cytotoxic activity, as well as computational calculations.

Synthesis, characterization, DFT study, DNA/BSA-binding affinity, and cytotoxicity of some dinuclear and trinuclear gold(III) complexes.

In this study, we have synthesized a series of dinuclear and trinuclear gold(III) complexes of the general formula [Au2(N-N)Cl6] (1-3) for dinuclear and [Au3(N-N)2Cl8]+ (4-6) for trinuclear compounds, respectively, in which N-N is a bidentate ligand (1,4-diaminobutane; 1,6-diaminohexane or 1,8-diaminooctane). These complexes were characterized by elemental analysis, molar conductivity, and spectroscopic techniques (IR, UV-Vis, 1H NMR, ESI-MS). We performed DFT calculations to get insight into the geometry of the studies complexes. DNA-binding studies were performed by UV-Vis spectrophotometry and fluorescence spectroscopy. The results of competitive reactions between gold(III) complexes and ethidium bromide (EB) towards DNA have shown that selected complexes can displace EB from DNA-EB adduct. In addition, these experiments confirm that polynuclear gold(III) complexes interact with DNA covalently or via intercalation. Furthermore, high values of binding constants of gold(III) complexes towards bovine serum albumin (BSA) protein indicate good binding affinity. In addition, redox stability of complexes in the presence of DNA/BSA was confirmed by cyclic voltammetry. Results of the interactions between gold(III) complexes with DNA/BSA were discussed in reference to molecular docking data obtain by Molegro virtual docker. The cytotoxic activity of synthesized gold(III) complexes was evaluated on human breast cancer cell line (MDA-MB-231), human colorectal cancer cell line (HCT-116), and normal human lung fibroblast cell line (MRC-5). All complexes dose-dependently reduced cancer and normal cells viabilities, with significant cytotoxic effects (IC50 < 25 µM) for trinuclear gold(III) complexes (4, 5) on HCT-116 cells.

Guidelines-Driven Educational Intervention Promotes Healthy Lifestyle Among Adolescents and Adults: A Serbian National Longitudinal Study.

Background and objectives: The effectiveness of short-term focused educational programs to change health behaviors across large populations seems to be poorly described so far. The main aim of the present study was to evaluate an age-specific 45-min educational program, designed in accordance with the current U.S. Department of Health and Human Services and U.S. Department of Agriculture dietary guidelines and physical activity (PA) guidelines, among adolescents and adults. Materials and Methods: We evaluated the health-promoting lifestyle habits by the Health-Promoting Lifestyle Profile (HPLP-II) at baseline and following 6⁻8 weeks post-education in a nationally representative sample of Serbian adolescents and adults (n = 3822). Results: The percentage of adolescents eating 3⁻5 servings of vegetables per day increased at follow-up (20.1% versus 23.1%, p = 0.001), with significantly more adolescents regularly reading food labels (from 12.2% at baseline to 14.2% at follow-up; p = 0.02). Taken together, mean HPLP-II scores in adolescents significantly improved for both diet (0.05 points; p < 0.0001) and PA (0.09 points; p < 0.0001), and for PA in adults (0.08 points; p < 0.0001). Hierarchical multiple regression analysis revealed that our model as a whole (including time of testing as a predictor variable, and age and gender as control variables) explained 3.0% of the variance in mean HPLP-II scores for diet (p = 0.942) and 3.0% for PA (p = 0.285) in adolescents, and 1.1% of the variance in HPLP-II scores for diet (p = 0.781) and 1.9% for PA (p = 0.075) in adults, respectively. Conclusions: It appears that a brief focused education can positively tackle unhealthy lifestyles in promoting good health in general population. Different modes of interactive communication used here appeared to strengthen participants' capacities for lifestyle changes.

New monofunctional platinum(II) and palladium(II) complexes: Studies of the nucleophilic substitution reactions, DNA/BSA interaction, and cytotoxic activity.

Four new complexes [Pd(H2LtBu)Cl]Cl (Pd1), [Pt(H2LtBu)Cl]Cl (Pt1), [Pd(Me2LtBu)Cl]Cl (Pd2) and [Pt(Me2LtBu)Cl]Cl (Pt2) (where H2LtBu = 2,6-bis(5-(tert-butyl)-1H-pyrazol-3-yl)pyridine and Me2LtBu = 2,6-bis(5-(tert-butyl)-1-methyl-1H-pyrazol-3-yl)pyridine) were synthesized and characterized by elemental microanalysis, IR, 1H NMR and ESI-MS methods. The reactivity of complexes towards thiourea (Tu), l-methionine (l-Met), l-cysteine (l-Cys) and guanosine-5'-monophosphate (5'-GMP) was investigated. The obtained order was established as follows: Tu > l-Cys > l-Met > 5'-GMP. Complexes Pd1 and Pt1, that contain H2LtBu as chelator, showed higher reactivity towards biomolecules than those with Me2LtBu. The interaction of complexes with calf thymus DNA (CT-DNA) and bovine serum albumin (BSA) was studied by UV-Vis and fluorescence spectroscopy. The results have shown that complexes can bind to DNA exhibiting high binding constants (Kb = 104 M-1). Obtained results during the examination of competitive reaction with ethidium bromide (EB) showed that complexes can replace EB-bound DNA. High values of binding constants indicate good binding affinity of complexes towards BSA. We evaluated the stability differences between complexes based on terpy as well as H2LtBu/Me2LtBu by DFT calculations (B3LYP(CPCM)/LANL2DZp), showing that both tridentate ligand systems lead to complexes of similar stability. The results of biological testing showed that all complexes exert moderate to high selective cytotoxicity, inducing apoptosis and autophagy in HeLa and PANC-1 tumor cell lines. Pd1 exhibited the strongest cytotoxic effect. Finally, cell cycle analysis showed that in HeLa cells Pd1, Pd2 and Pt1 induced accumulation of cells in S phase, whereas in PANC-1 cells Pd2 and Pt1 induced G2/M cycle arrest and Pd1 induced G0/G1 arrest.

New gold pincer-type complexes: synthesis, characterization, DNA binding studies and cytotoxicity.

With the aim of assessing whether Au(iii) compounds with pincer type ligands might be utilized as potential antitumor agents, three new monofunctional Au(iii) complexes of the general formula [Au(N-N'-N)Cl]Cl2, where N-N'-N = 2,6-bis(5-tert-butyl-1H-pyrazol-3-yl)pyridine (H2LtBu, 1), 2,6-bis(5-tert-butyl-1-methyl-1H-pyrazol-3-yl)pyridine (Me2LtBu, 2) or 2,6-bis((4S,7R)-1,7,8,8-tetramethyl-4,5,6,7-tetrahydro-1H-4,7-methanoindazol-3-yl)pyridine (Me2*L, 3) were synthesized. All complexes were characterized by elemental analysis, spectroscopic techniques (IR, UV-Vis, 1D and 2D NMR) and mass spectrometry (MALDI TOF MS). The chemical behavior of the complexes under physiological conditions was studied by UV-Vis spectroscopy, which showed that all compounds were remarkably stable and that the gold center remained in the 3+ oxidation state. The kinetics and the mechanism of the reaction of complexes 1-3 with guanine derivatives (i.e. guanosine (Guo) and guanosine-5'-monophosphate (5'-GMP)) and calf thymus DNA (CT DNA) were studied by stopped-flow spectroscopy. The three complexes displayed moderately different rate constants in their reactions with Guo, 5'-GMP and CT DNA, which can be explained by the steric hindrance and σ-donicity of the methyl substituent on the bis-pyrazolylpyridine fragment in complexes 2 and 3. The measured enthalpies and entropies of activation (ΔH≠ > 0, ΔS≠ < 0) supported an associative mechanism for the substitution process. The interaction of the newly synthesized complexes 1-3 with CT DNA was investigated by UV-Vis and fluorescence spectroscopy, and also by viscosity measurements, which all indicated that complexes 1-3 bound to CT DNA with moderate binding affinity (Kb = 1.6-5.7 × 103 M-1) and stabilized the duplex of CT DNA. Molecular docking indicated that complexes 1-3 interacted with DNA via intercalation. Complex 1 reduced the cell survival of all the investigated cell lines (A549, A375, and LS-174) with IC50 values being up to 20 µM. We have shown that 1 induced perturbations of the cell cycle and led to apoptosis in human melanoma A375 cells. Complex 1 also affected the level of reactive oxygen species (ROS) in the same cells. However, pre-treatment of A375 cells with NAC (ROS scavenger) reversed the effect of 1 on their survival.

Thinness in young schoolchildren in Serbia: another case of the double burden of malnutrition?

OBJECTIVE: Thinness is rarely highlighted or regularly monitored among children in developed countries although it may be rather frequent and pose a significant risk to children's health. We aimed to describe the prevalence of mild, moderate and severe thinness among young Serbian schoolchildren. DESIGN: Cross-sectional study of schoolchildren aged 6-9 years. Children were assessed for weight, height and BMI as part of the WHO European Childhood Obesity Surveillance Initiative in Serbia. Thinness grades were defined as gender- and age-specific cut-offs for BMI according to the International Obesity Task Force criteria. SETTING: Serbia, September to November 2015. SUBJECTS: Students (n 4861) in grades 2 and 3 (6-9 years, 2397 girls). RESULTS: Overall prevalence of thinness in Serbian schoolchildren was 9·6 %. Mild thinness was clearly the largest category with a prevalence of 7·6 %, moderate thinness was present in 1·7 % of children and severe thinness was found in 0·3 % of children. OR indicated a significant risk of being thin for girls (1·44 times higher compared with boys) and children attending schools with no health-focused educational programme (1·57 times more likely to be thin than peers enrolled in schools with such programmes). In addition, OR for thinness tended to be 1·23 times higher in children living in an economically disadvantaged region of Serbia (P=0·06). CONCLUSIONS: A rather high prevalence of thinness highlights this malnutrition disorder as an emerging health issue that should trigger public health policies to tackle thinness, especially in girls of young age and children living in economically disadvantaged areas.

Impact of aromaticity on anticancer activity of polypyridyl ruthenium(II) complexes: synthesis, structure, DNA/protein binding, lipophilicity and anticancer activity.

With the aim of assessing how the aromaticity of the inert chelating ligand can influence the activity of ruthenium(II) polypyridyl complexes, two new monofunctional ruthenium(II) complexes, [Ru(Cl-Ph-tpy)(phen)Cl]Cl (1) and [Ru(Cl-Ph-tpy)(o-bqdi)Cl]Cl (2) (where Cl-Ph-tpy = 4'-(4-chlorophenyl)-2,2':6',2″-terpyridine, phen = 1,10-phenanthroline, o-bqdi = o-benzoquinonediimine), were synthesized. All complexes were fully characterized by elemental analysis and spectroscopic techniques (IR, UV-Vis, 1D and 2D NMR, XRD). Their chemical behavior in aqueous solution was studied by UV-Vis and NMR spectroscopy showing that both compounds are relatively labile leading to the formation of the corresponding aqua species 1a and 2a. 1H NMR spectroscopy studies performed on complexes 1 and 2 demonstrated that after the hydrolysis of the Cl ligand, they are capable to interact with guanine derivatives (i.e., 9-methylguanine (9MeG) and 5'-GMP) through the N7, forming monofunctional adduct. The kinetics and the mechanism of the reaction of complexes 1 and 2 with the biologically more relevant 5'-GMP ligand were studied by UV-Vis spectroscopy. DNA/protein interactions of the complexes have been examined by photophysical studies, which demonstrated a bifunctional binding mode of the complexes with DNA and the complexes strongly quench the fluorescence intensity of bovine serum albumin (BSA) through the mechanism of both static and dynamic quenching. Complexes 1 and 2 strongly induced apoptosis of treated cancer cells with high percentages of apoptotic cells and negligible percentage of necrotic cells. In addition, both ruthenium complexes decreased Bcl-2/Bax ratio causing cytochrome c mitochondrial release, the activation of caspase-3 and induction of apoptosis.

WHO European Childhood Obesity Surveillance Initiative in Serbia: a prevalence of overweight and obesity among 6-9-year-old school children.

BACKGROUND: The World Health Organization (WHO) European Childhood Obesity Surveillance Initiative (COSI) is a public health program established in order to understand the progress of the obesity epidemic in young populations and gain inter-country comparisons within the European region, yet the data from a number of East European countries, including Serbia, were not available then. Therefore, the main aim of this cross-sectional study was to collect data about the prevalence of overweight and obesity among 6-9-year-old school children in Serbia according to the standardized protocol during the Fourth COSI Implementation Round. METHODS: From September 2015 to November 2015, 5102 first- and second-grade primary-school children (age 7.7±0.6 years) were assessed for weight, height, and body mass index (BMI) in 14 Serbian school districts. RESULTS: The prevalence rates of obesity, as calculated using the International Obesity Task Force (IOTF) cut-off points, vary across different age groups, with the lowest obesity rates reported in 7-year-old boys (6.2%), while the highest obesity prevalence rates were observed in 6-year-old boys (9.7%). In addition, being overweight was strongly associated with poor local community development and lower level of urbanization. The overall prevalence of overweight (23.1%, including obesity) and obesity (6.9%) in Serbian primary-school children seem to be comparable to rather high rates previously reported in other countries participating in the COSI program, indicating an obesity epidemic in Serbian children. CONCLUSIONS: This surveillance system should be regularly implemented throughout Europe, providing comparable data on rates of overweight/obesity in primary schools that might drive prudent actions to reverse the pandemic trend of childhood obesity.

[Structural and functional characteristics of urinary tract in offspring of Balkan endemic nephropathy patients].

INTRODUCTION: Balkan endemic nephropathy (BEN) is a familial chronic progressive tubulointerstitial disease of unknown aetiology that occurs with high prevalence in endemic rural environments of Serbia, Bosnia and Herzegovina, Croatia, Bulgaria and Romania. It has been documented only in adults. OBJECTIVE: The aim of this study was to examine clinical markers of BEN in children and adolescent offspring of BEN patients. METHODS: Prospective clinical trial involved two groups of children and adolescents: I consisted of 30 offspring of BEN patients and II of 29 offspring of non-BEN dialysis patients, both of them living in the same South Morava region of Serbia. All of them were healthy at the time of the investigation, not receiving any drugs. The study included personal and family history, physical examination, comprehensive laboratory analyses and renal ultrasound. Blood pressure (BP) was determined by using casual BP and 24 h ABPM in subjects older than 5 years. Urinary proteins were investigated by analysing microalbumin, alfa 1 microglobulin, beta 2 microglobulin and SDS-PAGE electrophoresis. GFR was measured by estimated creatinine clearance and by serum Cystatin C concentrations. RESULTS: There were no statistically significant differences in age, gender, history of urinary tract infections or functional voiding disorders between these two groups. All of the studied subjects had normal BP and GFR. Renal ultrasound was abnormal only in BEN offspring (6.66%) as well as increased urine concentrations of microalbumin (3.3%), alpha 1 microglobulin (10%) and beta 2 microglobulin (13.3%) while low molecular protein (<66,000 D) was prevalent in BEN compared with non-BEN offspring (21.43% vs. 3.7%). CONCLUSION: Renal abnormalities in offspring of BEN patients may be an early marker of BEN.This has to be confirmed in long term follow-up of a greater number of BEN paediatric offspring.

[Investigation of Balkan endemic nephropathy in Serbia: how to proceed?].

Balkan endemic nephropathy (BEN) presents an unsolved puzzle despite fifty years of its investigation. Academy of Medical Sciences of the Serbian Medical Society organized a round table discussion on current unsolved problems related to BEN. The present paper summarizes presentations, discussion and conclusions of this meeting. During the last fifty years, the course of BEN prolonged and it shifted towards the older age in all endemic foci. Data on the incidence of BEN have been controversial and frequently based on the data on the number of BEN patients starting haemodialysis treatment. In Serbia, BEN patients present 6.5% of haemodialysis population and this percentage differs among different centres ranging from 5% (Leskovac) to 46% (Lazarevac). Maintenance of high prevalence of BEN patients on regular haemodialysis indicates that BEN is not an expiring disease. In addition, recent data have shown more frequent microalbuminuria and low-molecular weight proteinuria in children from endemic than from nonendemic families. Aetiology of BEN is still unknown despite numerous investigations of environmental and genetic factors. Today, there is a very current hypothesis on the aetiological role of aristolochic acid but the role of viruses, geochemical factors and genetic factors must not be neglected. Morphological features of BEN are nonspecific and characterized by acellular interstitial fibrosis, tubular atrophy and changes on pre- and postglomerular vessels. New immunohistochemical and molecular biology methods offer a new approach to BEN investigation. Association of BEN with high incidence of upper-urothelial tumours is well-known. Recent studies have shown significant changes of demographic characteristics of patients suffering upper-urothelial tumours, their prevalence in different endemic foci and characteristics of tumours. Further studies of BEN should be directed to determination of incidence and prevalence of disease in different endemic foci, investigations of different insufficiently examined aetiological factors as well as pathomorphological features of the disease by the use of modern methods.