Characterization of the effects of thymol derivatives on colorectal cancer spheroids.

Colorectal cancer (CRC) is one of the most commonly diagnosed malignancies with a high mortality rate. In the last few years, attention has been focused on substances of natural origin with anticancer activity. One such substance is thymol and its derivatives, which have been shown to have an antitumor effect also against CRC cells. In our study, we focused on determining the biological and antibacterial effects of thymol and thymol derivatives. Analyses were performed on a 3D model of human colon carcinoma cell lines (HCT-116 and HT-29) - spheroids. The cytotoxic (MTT assay) and genotoxic effect (comet assay) of thymol and derivatives: acetic acid thymol ester and thymol ß-D-glucoside were determined. ROS levels (ROS-Glo™ H2O2 Assay) and total antioxidant status (Randox TAS Assay) were also monitored. Last but not least, we also detected the effect of the derivatives using a disk diffusion assay and determined the number of colonies on the plates on selected bacteria such as Lacticaseibacillus rhamnosus, Lactiplantibacillus plantarum, Lacticaseibacillus paracasei, Lactobacillus brevis, Lactobacillus pentosus and Weizmannia coagulans. The derivatives did not show a significant inhibitory effect on the growth of LAB bacteria (lactic acid bacteria) in contrast to thymol. Overall, thymol derivatives are cytotoxic, genotoxic and increase ROS levels. Among the derivatives tested, acetic acid thymol ester (IC50 ~ 0.2 µg/ml) was more effective. The second derivative tested (thymol ß-D-glucoside) was effective at higher concentrations than thymol. Our research confirmed that thymol derivatives have a toxic effect on the 3D model of intestinal tumor cells, while they do not have a toxic effect on selected intestinal bacteria. Thus, they could bring new significance to the prevention or treatment of CRC.

Negative Charge-Carrying Glycans Attached to Exosomes as Novel Liquid Biopsy Marker.

Prostate cancer (PCa) is the second most common cancer. In this paper, the isolation and properties of exosomes as potential novel liquid biopsy markers for early PCa liquid biopsy diagnosis are investigated using two prostate human cell lines, i.e., benign (control) cell line RWPE1 and carcinoma cell line 22Rv1. Exosomes produced by both cell lines are characterised by various methods including nanoparticle-tracking analysis, dynamic light scattering, scanning electron microscopy and atomic force microscopy. In addition, surface plasmon resonance (SPR) is used to study three different receptors on the exosomal surface (CD63, CD81 and prostate-specific membrane antigen-PMSA), implementing monoclonal antibodies and identifying the type of glycans present on the surface of exosomes using lectins (glycan-recognising proteins). Electrochemical analysis is used to understand the interfacial properties of exosomes. The results indicate that cancerous exosomes are smaller, are produced at higher concentrations, and exhibit more nega tive zeta potential than the control exosomes. The SPR experiments confirm that negatively charged α-2,3- and α-2,6-sialic acid-containing glycans are found in greater abundance on carcinoma exosomes, whereas bisecting and branched glycans are more abundant in the control exosomes. The SPR results also show that a sandwich antibody/exosomes/lectins configuration could be constructed for effective glycoprofiling of exosomes as a novel liquid biopsy marker.

Genome-wide DNA methylome and transcriptome changes induced by inorganic nanoparticles in human kidney cells after chronic exposure.

The unique physicochemical properties make inorganic nanoparticles (INPs) an exciting tool in diagnosis and disease management. However, as INPs are relatively difficult to fully degrade and excrete, their unintended accumulation in the tissue might result in adverse health effects. Herein, we provide a methylome-transcriptome framework for chronic effects of INPs, commonly used in biomedical applications, in human kidney TH-1 cells. Renal clearance is one of the most important routes of nanoparticle excretion; therefore, a detailed evaluation of nanoparticle-mediated nephrotoxicity is an important task. Integrated analysis of methylome and transcriptome changes induced by INPs (PEG-AuNPs, Fe3O4NPs, SiO2NPs, and TiO2NPs) revealed significantly deregulated genes with functional classification in immune response, DNA damage, and cancer-related pathways. Although most deregulated genes were unique to individual INPs, a relatively high proportion of them encoded the transcription factors. Interestingly, FOS hypermethylation inversely correlating with gene expression was associated with all INPs exposures. Our study emphasizes the need for a more comprehensive investigation of INPs' biological safety, especially after chronic exposure.

Newly Synthesized Thymol Derivative and Its Effect on Colorectal Cancer Cells.

Thymol affects various types of tumor cell lines, including colorectal cancer cells. However, the hydrophobic properties of thymol prevent its wider use. Therefore, new derivatives (acetic acid thymol ester, thymol ß-D-glucoside) have been synthesized with respect to hydrophilic properties. The cytotoxic effect of the new derivatives on the colorectal cancer cell lines HT-29 and HCT-116 was assessed via MTT assay. The genotoxic effect was determined by comet assay and micronucleus analysis. ROS production was evaluated using ROS-Glo™ H2O2 Assay. We confirmed that one of the thymol derivatives (acetic acid thymol ester) has the potential to have a cyto/genotoxic effect on colorectal cancer cells, even at much lower (IC50~0.08 µg/mL) concentrations than standard thymol (IC50~60 µg/mL) after 24 h of treatment. On the other side, the genotoxic effect of the second studied derivative-thymol ß-D-glucoside was observed at a concentration of about 1000 µg/mL. The antiproliferative effect of studied derivatives of thymol on the colorectal cancer cell lines was found to be both dose- and time-dependent at 100 h. Moreover, thymol derivative-treated cells did not show any significantly increased rate of micronuclei formation. New derivatives of thymol significantly increased ROS production too. The results confirmed that the effect of the derivative on tumor cells depends on its chemical structure, but further detailed research is needed. However, thymol and its derivatives have great potential in the prevention and treatment of colorectal cancer, which remains one of the most common cancers in the world.

Antioxidant, Cytotoxic, Genotoxic, and DNA-Protective Potential of 2,3-Substituted Quinazolinones: Structure-Activity Relationship Study.

The evaluation of antioxidant compounds that counteract the mutagenic effects caused by the direct action of reactive oxygen species on DNA molecule is of considerable interest. Therefore, a series of 2,3-substituted quinazolinone derivatives (Q1-Q8) were investigated by different assays, and the relationship between their biological properties and chemical structure was examined. Genotoxicity and the potential DNA-protective effects of Q1-Q8 were evaluated by comet assay and DNA topology assay. Antioxidant activity was examined by DPPH-radical-scavenging, reducing-power, and total antioxidant status (TAS) assays. The cytotoxic effect of compounds was assessed in human renal epithelial cells (TH-1) and renal carcinoma cells (Caki-1) by MTT assay. Analysis of the structure-activity relationship disclosed significant differences in the activity depending on the substitution pattern. Derivatives Q5-Q8, bearing electron-donating moieties, were the most potent members of this series. Compounds were not genotoxic and considerably decreased the levels of DNA lesions induced by oxidants (H2O2, Fe2+ ions). Furthermore, compounds exhibited higher cytotoxicity in Caki-1 compared to that in TH-1 cells. Substantial antioxidant effect and DNA-protectivity along with the absence of genotoxicity suggested that the studied quinazolinones might represent potential model structures for the development of pharmacologically active agents.

Are Changes in the Percentage of Specific Leukocyte Subpopulations Associated with Endogenous DNA Damage Levels in Testicular Cancer Patients?

Chemoresistance of germ cell tumors (GCTs) represents an intensively studied property of GCTs that is the result of a complicated multifactorial process. One of the driving factors in this process is the tumor microenvironment (TME). Intensive crosstalk between the DNA damage/DNA repair pathways and the TME has already been reported. This study aimed at evaluating the interplay between the immune TME and endogenous DNA damage levels in GCT patients. A cocultivation system consisting of peripheral blood mononuclear cells (PBMCs) from healthy donors and GCT cell lines was used in an in vitro study. The patient cohort included 74 chemotherapy-naïve GCT patients. Endogenous DNA damage levels were measured by comet assay. Immunophenotyping of leukocyte subpopulations was performed using flow cytometry. Statistical analysis included data assessing immunophenotypes, DNA damage levels and clinicopathological characteristics of enrolled patients. The DNA damage level in PBMCs cocultivated with cisplatin (CDDP)-resistant GCT cell lines was significantly higher than in PBMCs cocultivated with their sensitive counterparts. In GCT patients, endogenous DNA damage levels above the cutoff value were independently associated with increased percentages of natural killer cells, CD16-positive dendritic cells and regulatory T cells. The crosstalk between the endogenous DNA damage level and specific changes in the immune TME reflected in the blood of GCT patients was revealed. The obtained data contribute to a deeper understanding of ongoing interactions in the TME of GCTs.

The Effect of Ten Essential Oils on Several Cutaneous Drug-Resistant Microorganisms and Their Cyto/Genotoxic and Antioxidant Properties.

In this study, we determined the antimicrobial activity of ten essential oils (EOs)-oregano, thyme, clove, arborvitae, cassia, lemongrass, melaleuca, eucalyptus, lavender, and clary sage-against drug-resistant microorganisms previously isolated from patients with skin infections. The essential oil compositions were determined using gas chromatography coupled to mass spectrometry (GC/MS). The assayed bacteria included Pseudomonas aeruginosa, Proteus vulgaris, Citrobacter koseri, and Klebsiella pneumoniae. Two drug-resistant yeasts (Candida albicans and Candida parapsilosis) were also involved in our survey. Oregano, thyme, cassia, lemongrass and arborvitae showed very strong antibacterial and antifungal activity against all tested strains. These results show that these essential oils may be effective in preventing the growth of the drug-resistant microorganisms responsible for wound infections. In this study, the genotoxic effects of tested essential oils on healthy human keratinocytes HaCaT were evaluated using the comet assay for the first time. These results revealed that none of the essential oils induced significant DNA damage in vitro after 24 h. Moreover, the treatment of HaCaT cells with essential oils increased the total antioxidant status (TAS) level. The obtained results indicate that EOs could be used as a potential source of safe and potent natural antimicrobial and antioxidant agents in the pharmaceutical and food industries.

Hyperforin Exhibits Antigenotoxic Activity on Human and Bacterial Cells.

Hyperforin (HF), a substance that accumulates in the leaves and flowers of Hypericum perforatum L. (St. John's wort), consists of a phloroglucinol skeleton with lipophilic isoprene chains. HF exhibits several medicinal properties and is mainly used as an antidepressant. So far, the antigenotoxicity of HF has not been investigated at the level of primary genetic damage, gene mutations, and chromosome aberrations, simultaneously. The present work is designed to investigate the potential antigenotoxic effects of HF using three different experimental test systems. The antigenotoxic effect of HF leading to the decrease of primary/transient promutagenic genetic changes was detected by the alkaline comet assay on human lymphocytes. The HF antimutagenic effect leading to the reduction of gene mutations was assessed using the Ames test on the standard Salmonella typhimurium (TA97, TA98, and TA100) bacterial strains, and the anticlastogenic effect of HF leading to the reduction of chromosome aberrations was evaluated by the in vitro mammalian chromosome aberration test on the human tumor cell line HepG2 and the non-carcinogenic cell line VH10. Our findings provided evidence that HF showed antigenotoxic effects towards oxidative mutagen zeocin in the comet assay and diagnostic mutagen (4-nitroquinoline-1-oxide) in the Ames test. Moreover, HF exhibited an anticlastogenic effect towards benzo(a)pyrene and cisplatin in the chromosome aberration test.

Enriching the drinking water of rats with extracts of Salvia officinalis and Thymus vulgaris increases their resistance to oxidative stress.

Nature is an attractive source of therapeutic compounds. In comparison to the artificial drugs, natural compounds cause less adverse side effects and are suitable for current molecularly oriented approaches to drug development and their mutual combining. Medicinal plants represent one of the most available remedy against various diseases. Proper examples are Salvia officinalis L. and Thymus vulgaris L. which are known aromatic medicinal plants. They are very popular and frequently used in many countries. The molecular mechanism of their biological activity has not yet been fully understood. The aim of this study was to ascertain if liver cells of experimental animals drinking extracts of sage or thyme will manifest increased resistance against oxidative stress. Adult Sprague-Dawley rats were divided into seven groups. They drank sage or thyme extracts for 2 weeks. At the end of the drinking period, blood samples were collected for determination of liver biochemical parameters and hepatocytes were isolated to analyze (i) oxidatively generated DNA damage (conventional and modified comet assay), (ii) activities of antioxidant enzymes [superoxide dismutase (SOD), glutathione peroxidase (GPx)] and (iii) content of glutathione. Intake of sage and thyme had no effect either on the basal level of DNA damage or on the activity of SOD in rat hepatocytes and did not change the biochemical parameters of blood plasma. Simultaneously, the activity of GPx was significantly increased and the level of DNA damage induced by oxidants was decreased. Moreover, sage extract was able to start up the antioxidant protection expressed by increased content of glutathione. Our results indicate that the consumption of S.officinalis and T.vulgaris extracts positively affects resistency of rat liver cells against oxidative stress and may have hepatoprotective potential.

Comparison of biological processes induced in HepG2 cells by tert-butyl hydroperoxide (t-BHP) and hydroperoxide (H2O2): The influence of carvacrol.

This paper presents comparisons of biological impacts of the oxidants H2O2 and t-BHP on human liver cells, and shows modulation of these effects by the phenolic compound carvacrol. To understand better how these oxidants exert their effect on DNA and on the activity of the enzymes superoxide dismutase (SOD) and glutathione peroxidase (GPx), we measured intracellular antioxidant glutathione (iGSH) and intracellular reactive oxidative species (iROS). DNA lesions corresponded to single-strand DNA breaks, alkali-labile lesions and formamido-pyrimidine-DNA-glycosylase (FPG)-sensitive sites. Pre-treatment of cells with carvacrol substantially decreased the number of H2O2-induced DNA lesions, but the number of t-BHP-induced DNA lesions was not reduced. Activities of both SOD and GPx were stimulated significantly by carvacrol and were reduced by the combined effect of carvacrol and oxidants. H2O2 and t-BHP alone influenced the level of antioxidant enzymes differently. While H2O2 did not markedly change the activity of SOD or GPx, lower concentrations of t-BHP stimulated activity of SOD and mainly GPx. The level of iROS was increased by both oxidants and decreased by carvacrol applied either alone or with oxidants. The level of iGSH was not influenced in any of the treatments tested. Our results show that although both oxidants induced oxidative stress and damaged cellular DNA, their influences on other molecular processes were different. The protective effect of carvacrol against DNA-damaging effects of H2O2 was unambiguous, but reduction by carvacrol of the DNA-damaging effect of t-BHP was not observed. These results suggest that the phenolic compound carvacrol contributes to the defence mechanisms of the human organism, but these beneficial effects are dependent on the origin and source of the actual oxidative stress.

Rapid identification of in vitro cell toxicity using an electrochemical membrane screening platform.

This study compares the performance and output of an electrochemical phospholipid membrane platform against respective in vitro cell-based toxicity testing methods using three toxicants of different biological action (chlorpromazine (CPZ), colchicine (COL) and methyl methanesulphonate (MMS)). Human cell lines from seven different tissues (lung, liver, kidney, placenta, intestine, immune system) were used to validate this physicochemical testing system. For the cell-based systems, the effective concentration at 50 % cell death (EC50) values are calculated. For the membrane sensor, a limit of detection (LoD) value was extracted as a quantitative parameter describing the minimum concentration of toxicant which significantly affects the structure of the phospholipid sensor membrane layer. LoD values were found to align well with the EC50 values when acute cell viability was used as an end-point and showed a similar toxicity ranking of the tested toxicants. Using the colony forming efficiency (CFE) or DNA damage as end-point, a different order of toxicity ranking was observed. The results of this study showed that the electrochemical membrane sensor generates a parameter relating to biomembrane damage, which is the predominant factor in decreasing cell viability when in vitro models are acutely exposed to toxicants. These results lead the way to using electrochemical membrane-based sensors for rapid relevant preliminary toxicity screens.

Borneol administration protects primary rat hepatocytes against exogenous oxidative DNA damage.

Experimental evidences suggest that most essential oils possess a wide range of biological and pharmacological activities that may protect tissues against oxidative damage. In this study, we investigated DNA-protective effect of borneol, a component of many essential oils, against oxidative DNA damage induced in primary cultures of rat hepatocytes. Borneol was added to drinking water of Sprague-Dawley rats and DNA resistance against oxidative agents was compared in hepatocytes originated from control and borneol-treated rats. Oxidative stress induced by visible light-excited methylene blue (MB/VL) or 2,3-dimethoxy-1,4-naphthoquionone (DMNQ) resulted in increased levels of DNA lesions measured by the modified single cell gel electrophoresis. Borneol (17 or 34 mg/kg body weight) added to drinking water of rats for 7 days reduced the level of oxidative DNA lesions induced in their hepatocytes by MB/VL or DMNQ. To explain the increased resistance of DNA towards oxidative stress, we measured the base-excision repair (BER) capacity in liver cell extracts of control and borneol-supplemented rats on DNA substrate of HepG2 cells containing oxidative damage. Our results showed that administration of borneol in drinking water had no effect on incision activity of hepatocytes isolated from supplemented rats. The spectrophotometric assessment of enzymatic antioxidants superoxide dismutase (SOD) and glutathione peroxidase (GPx) activities and the flow cytometric assessment of total intracellular glutathione (iGSH) in primary hepatocytes of borneol-supplemented rats showed no changes in SOD and GPx activities but higher iGSH content particularly in hepatocytes of higher borneol dose (34 mg/kg) supplemented rats in comparison to control animals. Despite the fact that borneol had no effect either on BER of oxidative DNA damage or on the levels of antioxidant enzymes and manifested no reducing power and radicals scavenging activity, it increased significantly the level of non-enzymatic antioxidant iGSH which could reduce the oxidative DNA lesions induced by MB/VL or DMNQ.

Exosomes from prostate cancer cell lines: Isolation optimisation and characterisation.

Exosomes are considered to be a rich source of biomarkers, hence in this article we examine the best procedure for their isolation. We examine several isolation procedures, exosome storage conditions and other conditions affecting exosome production by prostate cell lines. We selected four different commercially available kits based on different principles to achieve exosome isolation, the best being magnetic-based. In addition, we found storage at - 20 °C to be good for storing isolated exosomes and that exosomes were produced from the cancerous prostate cell line 22Rv1 in much greater amounts than the non-cancerous prostate cell line RWPE1. We also found differences in the response of both cell lines in the production of exosomes as a result of stress, i.e. exposure to hydrogen peroxide and starvation. The effect of Triton X-100 on exosome lysis was examined using two different surfactant concentrations by analysis of the exosome count and change in the exosome size. The final part of the article details the advantages of the use of a 2D biochip prepared in-house over a commercially available 3D biochip for monitoring the interaction of exosomes via its surface receptors (CD63) with an immobilised ligand (anti-CD63 antibodies) using surface plasmon resonance. The final experiment shows the potential of lectin fluorescent microarrays for the analysis of glycans present in lysed exosomes.

Pharmacokinetics of PEGylated Gold Nanoparticles: In Vitro-In Vivo Correlation.

Data suitable for assembling a physiologically-based pharmacokinetic (PBPK) model for nanoparticles (NPs) remain relatively scarce. Therefore, there is a trend in extrapolating the results of in vitro and in silico studies to in vivo nanoparticle hazard and risk assessment. To evaluate the reliability of such approach, a pharmacokinetic study was performed using the same polyethylene glycol-coated gold nanoparticles (PEG-AuNPs) in vitro and in vivo. As in vitro models, human cell lines TH1, A549, Hep G2, and 16HBE were employed. The in vivo PEG-AuNP biodistribution was assessed in rats. The internalization and exclusion of PEG-AuNPs in vitro were modeled as first-order rate processes with the partition coefficient describing the equilibrium distribution. The pharmacokinetic parameters were obtained by fitting the model to the in vitro data and subsequently used for PBPK simulation in vivo. Notable differences were observed in the internalized amount of Au in individual cell lines compared to the corresponding tissues in vivo, with the highest found for renal TH1 cells and kidneys. The main reason for these discrepancies is the absence of natural barriers in the in vitro conditions. Therefore, caution should be exercised when extrapolating in vitro data to predict the in vivo NP burden and response to exposure.

Electrospun Poly(ethylene Terephthalate)/Silk Fibroin Composite for Filtration Application.

In this study, fibrous membranes from recycled-poly(ethylene terephthalate)/silk fibroin (r-PSF) were prepared by electrospinning for filtration applications. The effect of silk fibroin on morphology, fibers diameters, pores size, wettability, chemical structure, thermo-mechanical properties, filtration efficiency, filtration performance, and comfort properties such as air and water vapor permeability was investigated. The filtration efficiency (FE) and quality factor (Qf), which represents filtration performance, were calculated from penetration through the membranes using aerosol particles ranging from 120 nm to 2.46 µm. The fiber diameter influenced both FE and Qf. However, the basis weight of the membranes has an effect, especially on the FE. The prepared membranes were classified according to EN149, and the most effective was assigned to the class FFP1 and according to EN1822 to the class H13. The impact of silk fibroin on the air permeability was assessed. Furthermore, the antibacterial activity against bacteria S. aureus and E. coli and biocompatibility were evaluated. It is discussed that antibacterial activity depends not only on the type of used materials but also on fibrous membranes' surface wettability. In vitro biocompatibility of the selected samples was studied, and it was proven to be of the non-cytotoxic effect of the keratinocytes (HaCaT) after 48 h of incubation.

Biofilm inhibition by biocompatible poly(ε-caprolactone) nanocapsules loaded with essential oils and their cyto/genotoxicity to human keratinocyte cell line.

Essential oils (EOs) of Thymus capitatus (Th) carvacrol chemotype and Origanum vulgare (Or) thymol and carvacrol chemotype were encapsulated in biocompatible poly(ε-caprolactone) nanocapsules (NCs). These nanosystems exhibited antibacterial, antifungal, and antibiofilm activities against Staphylococcus aureus, Escherichia coli, and Candida albicans. Th-NCs and Or-NCs were more effective against all tested strains than pure EOs and at the same time were not cytotoxic on HaCaT (T0020001) human keratinocyte cell line. The genotoxic effects of EO-NCs and EOs on HaCaT were evaluated using an alkaline comet assay for the first time, revealing that Th-NCs and Or-NCs did not induce DNA damage compared with untreated control HaCaT cells in vitro after 24 h. The cells morphological changes were assessed by label-free live cell Raman imaging. This study demonstrate the ability of poly(ε-caprolactone) nanocapsules loaded with thyme and oregano EOs to reduce microbial and biofilm growth and could be an ecological alternative in the development of new antimicrobial strategies.

Pharmacokinetics, Biodistribution, and Biosafety of PEGylated Gold Nanoparticles In Vivo.

Despite the obvious advantages of gold nanoparticles for biomedical applications, controversial and incomplete toxicological data hamper their widespread use. Here, we present the results from an in vivo toxicity study using gold nanoparticles coated with polyethylene glycol (PEG-AuNPs). The pharmacokinetics and biodistribution of PEG-AuNPs were examined in the rat's liver, lung, spleen, and kidney after a single i.v. injection (0.7 mg/kg) at different time intervals. PEG-AuNPs had a relatively long blood circulation time and accumulated primarily in the liver and spleen, where they remained for up to 28 days after administration. Increased cytoplasmic vacuolation in hepatocytes 24 h and 7 days after PEG-AuNPs exposure and apoptotic-like cells in white splenic pulp 24 h after administration has been detected, however, 28 days post-exposure were no longer observed. In contrast, at this time point, we identified significant changes in lipid metabolism, altered levels of liver injury markers, and elevated monocyte count, but without marked biological relevance. In blood cells, no DNA damage was present in any of the studied time intervals, with the exception of DNA breakage transiently detected in primary kidney cells 4 h post-injection. Our results indicate that the tissue accumulation of PEG-AuNPs might result in late toxic effects.

DNA damage response and preleukemic fusion genes induced by ionizing radiation in umbilical cord blood hematopoietic stem cells.

There is clear evidence that ionizing radiation (IR) causes leukemia. For many types of leukemia, the preleukemic fusion genes (PFG), as consequences of DNA damage and chromosomal translocations, occur in hematopoietic stem and progenitor cells (HSPC) in utero and could be detected in umbilical cord blood (UCB) of newborns. However, relatively limited information is available about radiation-induced apoptosis, DNA damage and PFG formation in human HSPC. In this study we revealed that CD34+ HSPC compared to lymphocytes: (i) are extremely radio-resistant showing delayed time kinetics of apoptosis, (ii) accumulate lower level of endogenous DNA damage/early apoptotic γH2AX pan-stained cells, (iii) have higher level of radiation-induced 53BP1 and γH2AX/53BP1 co-localized DNA double stranded breaks, and (iv) after low dose of IR may form very low level of BCR-ABL PFG. Within CD34+ HSPC we identified CD34+CD38+ progenitor cells as a highly apoptosis-resistant population, while CD34+CD38- hematopoietic stem/multipotent progenitor cells (HSC/MPP) as a population very sensitive to radiation-induced apoptosis. Our study provides critical insights into how human HSPC respond to IR in the context of DNA damage, apoptosis and PFG.

Nephrotoxicity: Topical issue.

Drug-induced kidney injury is one of the most significant adverse events and dose limiting factor in chemotherapy as well a major cause of prospective drug attrition during pharmaceutical development. Moreover, kidney injury can also occur as a consequence of exposures to environmental xenobiotics such as heavy metals, fungal toxins and nanomaterials. The lack of adequate in vitro human kidney models that mimic more realistically the in vivo conditions and the absence of suitable and robust, cost-effective and predictive cell-based in vitro assays contribute to an underestimation of the kidney toxic potential of new drugs and xenobiotics. Therefore, a rapid screening system capable to detect potential nephrotoxicity at early stages of drug discovery is an urgent need. Here we provide an overview of human cell lines currently used as a surrogate in vitro kidney models in nephrotoxicity studies, including their advantages and limitations. In addition, the capacity of the single cell gel electrophoresis (SCGE)/comet assay as a potential tool in kidney toxicants screening is discussed. Despite a limited number of studies using the comet assay to evaluate the drug-induced kidney damage potential, a considerable variability in SCGE methodology (e.g. lysis, unwinding, and electrophoresis conditions) has been observed. Before the comet assay can be included in nephrotoxicity testing, a basic guideline has to be developed. To test its feasibility, additional in vitro experiments including inter-laboratory validation studies based on this guideline have to be performed.

Microwaves from mobile phone induce reactive oxygen species but not DNA damage, preleukemic fusion genes and apoptosis in hematopoietic stem/progenitor cells.

Exposure to electromagnetic fields (EMF) has been associated with the increased risk of childhood leukemia, which arises from mutations induced within hematopoietic stem cells often through preleukemic fusion genes (PFG). In this study we investigated whether exposure to microwaves (MW) emitted by mobile phones could induce various biochemical markers of cellular damage including reactive oxygen species (ROS), DNA single and double strand breaks, PFG, and apoptosis in umbilical cord blood (UCB) cells including CD34+ hematopoietic stem/progenitor cells. UCB cells were exposed to MW pulsed signals from GSM900/UMTS test-mobile phone and ROS, apoptosis, DNA damage, and PFG were analyzed using flow cytometry, automated fluorescent microscopy, imaging flow cytometry, comet assay, and RT-qPCR. In general, no persisting difference in DNA damage, PFG and apoptosis between exposed and sham-exposed samples was detected. However, we found increased ROS level after 1 h of UMTS exposure that was not evident 3 h post-exposure. We also found that the level of ROS rise with the higher degree of cellular differentiation. Our data show that UCB cells exposed to pulsed MW developed transient increase in ROS that did not result in sustained DNA damage and apoptosis.