Abstract Clinopodium alpinum subsp. orontium (K.Malý) Govaerts and Thymus bracteosus Vis. ex Benth. are endemic Lamiaceae species in Bosnia and Herzegovina with rather limited data about their cytotoxic and genotoxic effects. This study aimed to analyse phenolic compounds composition of C. alpinum subsp. orontium and T. bracteosus aqueous and dimethyl sulphoxide (DMSO) extracts and their cytotoxic and genotoxic potential in human peripheral blood lymphocyte cultures. Among 33 analytes, 17 were identified and quantified in the examined extracts with the rosmarinic and chlorogenic acids as main constituents. Genotoxic effects of extracts from both species are proven at the highest applied dose. T. bracteosus extracts and DMSO as a solvent exhibited stronger genotoxic potential that should be further investigated in tumour cell lines. Nevertheless, non-endemic species with similar phenolic composition and bioactivity should be the first choice for medicinal purposes. Graphical Abstract

Abstract The aim of this study was to determine the cytogenotoxic effects of methylparaben, ethylparaben and butylparaben using battery of tests in plant cells (Allium cepa assay) and human lymphocytes (chromosome aberration test and alkaline comet assay). Our results for A. cepa assay showed that none of the tested parabens showed an inducing effect on root growth. Mitotic index values decreased with increasing parabens concentration. Ethylparaben (0.10 mg/L) induced a higher number of vagrants and multipolarity, as well as the number of sticky chromosomes (0.50 mg/L), while butylparaben (0.25 and 0.50 mg/L) increased the frequency of sticky chromosomes. Higher frequency of apoptosis and necrosis was observed for ethylparaben (0.50 mg/L) and methylparaben (0.10 and 0.50 mg/L). As for chromosome aberrations test in human lymphocytes, the mitotic index was reduced with an increase in the concentration of all three tested parabens. Differences between methylparaben (0.25 mg/L), ethylparaben (0.10 mg/L) and butylparaben (0.25 mg/L) and controls for acentric fragments, chromatid breaks and polyploidy were observed. Increased frequency of apoptosis was induced by methylparaben and ethylparaben at concentrations of 0.25 and 0.50 mg/L. Alkaline comet assay demonstrated that 0.25 and 0.50 mg/L of ethylparaben and butylparaben have genotoxic potential by increasing the tail intensity against controls. These results suggest that methyl-, ethyl- and butylparaben possess certain geno/cytotoxic potential.

Abstract Twelve previously synthesized, biologically active 2,6,7-trihydroxyxanthen-3-one derivatives were evaluated in vitro for antiproliferative activity. Compounds were screened against HeLa, SW620, HepG2 and A549 tumor cell lines. Compound with the trifluormethyl group on C-4’ position of the phenyl ring showed the best inhibitory activity towards HeLa and A549 tumor cells with IC50 of 0.7 and 4.1 µmol L−1, resp. Compound with chlorine and fluorine substituents on aryl ring showed the best antiproliferative activity against SW620 with IC50 of 4.1 µmol L–1 and against HepG2 tumor cell line with IC50 of 4.2 µmol L–1. Analyses of cytotoxic and genotoxic potential of the trifluormethyl derivative were performed with cytokinesis-block micronucleus cytome assay in human lymphocyte culture and revealed no genotoxic and cytotoxic effects. The most potent compounds were subjected to molecular docking simulations in order to analyse bindings to molecular targets and, at the same time, further support the results of experimental cytotoxic tests. Docking studies showed sites of importance in forming hydrogen bonds of the most potent compounds with targets of interest.

Inducing cell death in tumor cells has been recognized as a promising strategy in curing tumors. Parallely, natural products, especially those with long-known usage in folk medicine, are gaining demanding and extensive clinical interests. Aiming to contribute to overall knowledge of curcumin and luteolin antitumour potentials, we analyzed their effects on cell death induction in NFS-60 cell line, using Trypan blue exclusion assay and TransDetect® Anenexin V-EGFP/PI assay. Results show that both tested agents induce cell death, especially in higher applied concentrations, but further investigations are needed to elucidate the mechanisms behind it.

Abstract Plant bioflavonoids are widely present in the human diet and have various protective properties. In this study, we have demonstrated the capacity of delphinidin and luteolin to increase human telomerase reverse transcriptase (hTERT) expression level and act as protective agents against halogenated boroxine-induced genotoxic damage. Halogenated boroxine K2(B3O3F4OH) (HB), is a novel compound with potential for the treatment of both benign and malignant skin changes. In vivo and in vitro studies have confirmed the inhibitory effects of HB on carcinoma cell proliferation and cell cycle progression as well as enzyme inhibition. However, minor genotoxic effects of HB are registered in higher applied concentrations, but those can be suppressed by in vitro addition of delphinidin and luteolin in appropriate concentrations. Fresh peripheral blood samples were cultivated for 72 h followed by independent and concomitant treatments of HB with luteolin or delphinidin. We analyzed the differences in relative hTERT expression between series of treatments compared with controls, which were based on normalized ratios with housekeeping genes. The obtained results have shown that selected bioflavonoids induce upregulation of hTERT that may contribute to the repair of genotoxic damage in vitro.

Micromeria pulegium (Rochel) Benth. is an endemic species of Lamiacea family that includes frequently used plants in culinary and folk medicine. As cytotoxic potential of some species of Micromeria genus has been confirmed, this study aimed to test unknown antiproliferative and genotoxic potential of M. pulegium, endemic bh species, aqueous leaf extract in normal (human lymphocytes) and cancer (human melanoma GR-M) cells in order to protect small populations of native M. pulegium populations or promote its controlled micropropagation or cultivation. Cytokinesis-block micronucleus cytome assay was applied for human lymphocyte cultures, while trypan blue exclusion assay was used for evaluation of cytotoxicity in human GR-M melanoma cells. Results demonstrate no genotoxic effects up to concentration of 0.2 mg/ml in human lymphocyte in vitro but significant reduction of cell viability in human GR-M melanoma cell line cultures treated with 0.3 mg/ml of Micromeria extract.

Satureja subspicata and S. horvatii are endemic species of the Balkan Peninsula and often used in traditional medicine in Bosnia and Herzegovina to treat different health conditions. We aimed to analyze the unevaluated apoptotic, genotoxic and cytotoxic effects of two Satureja species, as well as their content of phenolics that are mainly responsible for the plant's biological activity. Apoptotic and geno/cytotoxic activities of S. subspicata and S. horvatii were investigated in vitro in human lymphocyte culture and in vivo in mice. The content of the main phenolics in plant extracts was determined by ultra-high pressure liquid chromatography-MS-MS (UHPLC–MS/MS). Genotoxic and cytotoxic activities of Satureja extracts were evaluated in vitro by applying a cytokinesis-block micronucleus cytome assay in human lymphocyte culture and in vivo applying a mice reticulocytes micronucleus assay. SALSA RT-MLPA R011-C1 apoptosis assay was used for measuring the relative expression of 44 genes associated with the regulation of the apoptotic pathways in human lymphocyte cultures treated with different concentrations of two Satureja extracts. The first analysis of phenolic compounds in S. horvatii and S. subspicata determined by an UHPLC-MS/MS method revealed high levels of rosmarinic and caffeic acids. Minor genotoxic potential was determined in relation to the tested concentrations while no cytostatic and cytotoxic effects were revealed in vitro. However, when applied in concentrations of 200 mg/kg per os, aqueous extracts of two Satureja species significantly decreased frequency of reticulocytes micronuclei in treated mice against controls. Extracts of S. subspicata and S. horvatii in concentrations of 0.2 mg/mL, regardless of solvent used, downregulated pro-apoptotic and upregulated anti-apoptotic genes, showing anti-apoptotic activity. Our results indicate that the registered anti-genotoxic and anti-apoptotic activity is most likely related to the high level of phenolic acids (particularly rosmarinic and caffeic) in the tested extracts.

Tartrazine (E 102) is widely used yellow food colorant. It is used in nonalcoholic and sports drinks, spicy chips, jams, jelly and chewing gum and also found in many non-food products like soaps, cosmetics, shampoo, vitamins and some drugs. Tartrazine belongs to the most important and diverse group of synthetic dyes – azo dyes. Their use often creates controversies in the public since some of them are toxic, carcinogenic, mutagenic and cause different disorders or allergic reactions. In this study we aimed to evaluate genotoxic potential of tartrazine in human lymphocytes culture and its cytotoxic potential in human lymphocytes and melanoma GR-M cell line. For testing of its genotoxic and cytotoxic potential in human lymphocyte culture, we used chromosome aberration analysis and cytokinesis-block micronucleus cytome assay. For the analysis of its cytotoxic potential in human melanoma cell culture, we applied trypan blue exclusion assay.

Genotoxic and cytotoxic effects of curcumin and sunset yellow were tested by the chromosome aberration analysis and cytokinesis-block micronucleus cytome assay in human lymphocyte culture. Water solutions of food dyes, in concentrations of 1, 2, 4 and 8 mM, were added to the cultures at the beginning of the cultivation period. Concentrations of 4 and 8 mM of sunset yellow induced significant increase in frequencies of cells with chromosome aberrations. Tested concentrations of sunset yellow significantly associated with frequencies of structural aberrations, chromatid-type aberrations, total aberrant cells and micronuclei showing considerable dose dependent clastogenic activity. In higher analyzed concentrations, curcumin significantly increased only nuclear buds frequency, suggesting its potential genotoxicity, while sunset yellow showed dose-dependent genotoxic potential. Obtained results point toward favorization of natural coloring agents in food consumption and emphasize the need of controlled use of food colorants.

Non-small cell lung cancer has been shown to be resistant to many forms of chemotherapy and is amongst the deadliest cancers worldwide. The antiproliferative effects of halogenated boroxine K2(B3O3F4OH), have been confirmed in multiple cancer cell lines including melanoma and breast cancer. The potential for this chemical treatment on non-small cell lung cancer cells was studied and the lower threshold concentration with the clear biological effect of halogenated boroxine, was determined. Appling MTT assays and the relative gene expression analysis of two genes of interest, RRBP1 and PER1, novel knowledge on the biological potential of halogenated boroxine (HB) was gained, but did not lead to biological explanations of the mechanisms of halogenated boroxine activity. The results of MTT assay showed a significant HB effect on non-small cell lung cancer in concentration of 0.5 mg/mL, while relative expression levels of RRBP1 and PER1 did not significantly differ regardless the concentration applied.

Abstract Recently it was found that dipotassium-trioxohydroxytetrafluorotriborate, K2(B3O3F4OH), is a potent and highly specific inhibitor of precancerous cell processes. We conducted gene expression profiling of human melanoma cells before and after treatment with two concentrations (0.1 and 1 mM) of this boron inorganic derivative in order to assess its effects on deregulation of genes associated with tumor pathways. Parallel trypan blue exclusion assay was performed to assess the cytotoxicity effects of this chemical. Treatment with K2(B3O3F4OH) induced a significant decrease of cell viability in melanoma cellline at both tested concentrations. Furthermore, these treatments caused deregulation of more than 30 genes known as common anti-tumor drug targets. IGF-1 and hTERT were found to be significantly downregulated and this result may imply potential use of K2(B3O3F4OH) as an inhibitor or human telomerase and insulin-like growth factor 1, both of which are associated with various tumor pathways.

Introduction: Cigarette smoking is associated with severe health problems, especially cancers. In addition, cigarette smoking causes different genotoxic effects. Chromosome aberrations are one of well-known intermediate end points in carcinogenesis. The aim of this study was to compare frequencies of chromosome aberrations in peripheral blood lymphocytes between young smokers and non-smokes groups.Methods: The study was conducted with 30 smokers (average age 26.93 years) and 30 non-smokers (average age 26.96 years), and included the analysis of 100 metaphases per each blood sample. Differences in the arithmetic means of determined frequencies of chromosome aberrations were tested by two-tailed t-test for independent samples with the significance level of p < 0.05.Results: The results showed a significant increase in the frequencies of chromatid-type aberrations and total structural chromosome aberrations in smoker group. Frequencies of numerical aberrations did not differ significantly between two groups.Conclusions: This study confirmed genotoxicity of cigarette smoking and provided new evidence about its clastogenic activity.