Comparison of Cytotoxic, Genotoxic, and DNA-Protective Effects of Skyrin on Cancerous vs. Non-Cancerous Human Cells.

Secondary metabolites as a potential source of anticancer therapeutics have been the subject of many studies. Since hypericin, a metabolite isolated from Hypericum perforatum L., shows several biomedical properties applicable in oncology, the aim of our study was to investigate its potential precursor skyrin in terms of genotoxic and DNA-protective effects. These skyrin effects were analyzed by cell-free methods, and cytotoxicity was estimated by an MTT assay and by a trypan blue exclusion test, while the genotoxic/antigenotoxic potential was examined by comet assay using non-cancerous human lymphocytes and the HepG2 cancer cell line. Skyrin did not show DNA-damaging effects but rather exhibited DNA-protectivity using a DNA-topology assay. However, we observed only weak antioxidant and chelating skyrin properties in other cell-free methods. Regarding the cytotoxic activity of skyrin, HepG2 cells were more prone to skyrin-induced death in comparison to human lymphocytes. Skyrin in non-cytotoxic concentrations did not exhibit elevated genotoxicity in both cell types. On the other hand, skyrin displayed moderate DNA-protective effects that were more noticeable in the case of non-cancerous human lymphocytes. The potential genotoxic effects of skyrin were not observed, and its DNA-protective capacity was more prominent in non-cancerous cells. Therefore, skyrin might be a promising agent used in anticancer therapy.

Imaging flow cytometry and fluorescence microscopy in assessing radiation response in lymphocytes from umbilical cord blood and cancer patients.

DNA double strand breaks (DSB) induced by ionizing radiation (IR) are usually measured using γH2AX/53BP1 DNA repair foci, that is considered to be the most sensitive assay for DSB analysis. While fluorescence microscopy (FM) is the gold standard for this analysis, imaging flow cytometry (IFC) may offer number of advantages such as lack of the fluorescence background, higher number of cells analyzed, and higher sensitivity in detection of DNA damage induced by IR at low doses. Along with appearance of γH2AX foci, the variable fraction of the cells exhibits homogeneously stained γH2AX signal resulting in so-called γH2AX pan-staining, which is believed to appear at early stages of apoptosis. Here, we investigated incidence of γH2AX pan-staining at different time points after irradiation with γ-rays using IFC and compared the obtained data with the data from FM. Appearance of γH2AX pan-staining during the apoptotic process was further analyzed by fluorescence-activated cell sorting (FACS) of cells at different stages of apoptosis and subsequent immunofluorescence analysis. Our results show that IFC was able to reveal dose dependence of pan-staining, while FM failed to detect all pan-staining cells. Moreover, we found that γH2AX pan-staining could be induced by therapeutic, but not low doses of γ-rays and correlate well with percentage of apoptotic cells was analyzed using flow cytometric Annexin-V/7-AAD assay. Further investigations showed that γH2AX pan-staining is formed in the early phases of apoptosis and remains until later stages of apoptotic process. Apoptotic DNA fragmentation as detected with comet assay using FM correlated with the percentage of live and late apoptotic/necrotic cells as analyzed by flow cytometry. Lastly, we successfully tested IFC for detection of γH2AX pan-staining and γH2AX/53BP1 DNA repair foci in lymphocyte of breast cancer patients after radiotherapy, which may be useful for assessing individual radiosensitivity in a clinically relevant cohort of patients.

Synthesis of Tyrosol and Hydroxytyrosol Glycofuranosides and Their Biochemical and Biological Activities in Cell-Free and Cellular Assays.

Tyrosol (T) and hydroxytyrosol (HOT) and their glycosides are promising candidates for applications in functional food products or in complementary therapy. A series of phenylethanoid glycofuranosides (PEGFs) were synthesized to compare some of their biochemical and biological activities with T and HOT. The optimization of glycosylation promoted by environmentally benign basic zinc carbonate was performed to prepare HOT α-L-arabino-, ß-D-apio-, and ß-D-ribofuranosides. T and HOT ß-D-fructofuranosides, prepared by enzymatic transfructosylation of T and HOT, were also included in the comparative study. The antioxidant capacity and DNA-protective potential of T, HOT, and PEGFs on plasmid DNA were determined using cell-free assays. The DNA-damaging potential of the studied compounds for human hepatoma HepG2 cells and their DNA-protective potential on HepG2 cells against hydrogen peroxide were evaluated using the comet assay. Experiments revealed a spectrum of different activities of the studied compounds. HOT and HOT ß-D-fructofuranoside appear to be the best-performing scavengers and protectants of plasmid DNA and HepG2 cells. T and T ß-D-fructofuranoside display almost zero or low scavenging/antioxidant activity and protective effects on plasmid DNA or HepG2 cells. The results imply that especially HOT ß-D-fructofuranoside and ß-D-apiofuranoside could be considered as prospective molecules for the subsequent design of supplements with potential in food and health protection.

Non-Thermal Plasma-A New Green Priming Agent for Plants?

Since the earliest agricultural attempts, humankind has been trying to improve crop quality and yields, as well as protect them from adverse conditions. Strategies to meet these goals include breeding, the use of fertilisers, and the genetic manipulation of crops, but also an interesting phenomenon called priming or adaptive response. Priming is based on an application of mild stress to prime a plant for another, mostly stronger stress. There are many priming techniques, such as osmopriming, halopriming, or using physical agents. Non-thermal plasma (NTP) represents a physical agent that contains a mixture of charged, neutral, and radical (mostly reactive oxygen and nitrogen species) particles, and can cause oxidative stress or even the death of cells or organisms upon interaction. However, under certain conditions, NTP can have the opposite effect, which has been previously documented for many plant species. Seed surface sterilization and growth enhancement are the most-reported positive effects of NTP on plants. Moreover, some studies suggest the role of NTP as a promising priming agent. This review deals with the effects of NTP treatment on plants from interaction with seed and cell surface, influence on cellular molecular processes, up to the adaptive response caused by NTP.

Genotoxic Effects of Tributyltin and Triphenyltin Isothiocyanates, Cognate RXR Ligands: Comparison in Human Breast Carcinoma MCF 7 and MDA-MB-231 Cells.

The cytotoxicity of two recently synthesized triorganotin isothiocyanate derivatives, nuclear retinoid X receptor ligands, was tested and compared in estrogen-receptor-positive MCF 7 and -negative MDA-MB-231 human breast carcinoma cell lines. A 48 h MTT assay indicated that tributyltin isothiocyanate (TBT-ITC) is more cytotoxic than triphenyltin isothiocyanate (TPT-ITC) in MCF 7 cells, and the same trend was observed in the MDA-MB-231 cell line. A comet assay revealed the presence of both crosslinks and increasing DNA damage levels after the 17 h treatment with both derivatives. Differences in cytotoxicity of TBT-ITC and TPT-ITC detected by FDA staining correspond to the MTT data, communicating more pronounced effects in MCF 7 than in the MDA-MB-231 cell line. Both derivatives were found to cause apoptosis, as shown by the mitochondrial membrane potential (MMP) depolarization and caspase-3/7 activation. The onset of caspase activation correlated with MMP dissipation and the total cytotoxicity more than with the amount of active caspases. In conclusion, our data suggest that the DNA damage induced by TBT-ITC and TPT-ITC treatment could underlie their cytotoxicity in the cell lines studied.

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.

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.

In vitro antiproliferative and cytotoxic activities of novel triphenyltin isoselenocyanate in human breast carcinoma cell lines MCF 7 and MDA-MB-231.

Intensive investigation for novel antiproliferative and cytotoxic effective chemical compounds is currently concentrated on structurally modified agents of natural or synthetic source. The selenium derivative of triorganotin compound, triphenyltin isoselenocyanate (TPT-NCSe) caused higher cytotoxicity in hormone sensitive MCF 7 (IC 50-250 nM) in comparison with triple-negative MDA-MB-231 breast carcinoma cell line (IC 50-450 nM) as determined by MTT assay. Measurement of DNA damage showed presence of crosslinks in both cell lines treated by increasing TPT-NCSe concentrations. This compound decreased mitochondrial membrane potential shown by JC-1 staining in a concentration-dependent manner in both cell lines. Activation of caspases-3/7 was observed in MDA-MB-231 cells and was significant only by concentrations causing significant level of crosslinks. On the other hand, migration assay revealed inhibitory effect of viability keeping 100 nM concentration of TPT-NCSe on migration of MDA-MB-231 cells. Our data has shown that this selenium containing triorganotin molecule exerts DNA damage-linked antineoplastic activity in breast carcinoma cell lines studied.

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.

Investigation of anti-oxidative, cytotoxic, DNA-damaging and DNA-protective effects of plant volatiles eugenol and borneol in human-derived HepG2, Caco-2 and VH10 cell lines.

Plant volatiles, which can get into the human organism in food, medicines, or cosmetic preparations, frequently manifest antibacterial, antifungal, antiviral and other effects. We studied anti-oxidative, cytotoxic, genotoxic and possible DNA-protective effects of eugenol and borneol. Anti-oxidative activities of aqueous and ethanolic solutions of these two volatile compounds of plants were determined by a spectrophotometric method by the use of the stable DPPH radical. Borneol did not show any anti-oxidative activity even at the highest concentrations soluble in water or ethanol (<1000mM), while eugenol did manifest anti-oxidative activity, and at much lower concentrations (5-100 microM). The cytotoxicity of eugenol and borneol as well as their DNA-damaging effects and their influence on sensitivity of cells against the DNA-damaging effects of H(2)O(2) were investigated in three different cell lines, i.e. malignant HepG2 hepatoma cells, malignant Caco-2 colon cells, and nonmalignant human VH10 fibroblasts. The trypan-blue exclusion assay showed that in the three cell lines the cytotoxicity of eugenol was significantly higher than that of borneol. Single-cell gel electrophoresis revealed that borneol did not cause any DNA strand-breaks at the concentrations studied, but showed that all concentrations of eugenol (<600 microM) significantly increased the level of DNA breaks in human VH10 fibroblasts and to a lower degree in Caco-2 colon cells. The DNA-damaging effects of eugenol were not observed in metabolically active HepG2 hepatoma cells. Borneol and eugenol differed also with respect to their DNA-protective effects. While borneol protected HepG2 and, to a lesser extent, VH10 cells (but not Caco-2) against H(2)O(2)-induced DNA damage, eugenol either did not change the cellular sensitivity to H(2)O(2) (HepG2 cells) or it even increased the sensitivity (Caco-2 and VH10 cells). These results do not indicate any correlation between the DNA-protective and the anti-oxidative capacities of eugenol and borneol.

Triorganotin Isothiocyanates Affect Migration and Immune Check-point Receptors in Human Triple-negative Breast Carcinoma MDA-MB-231 Cells.

BACKGROUND/AIM: Triple-negative breast cancer (TNBC) constitutes 15-20% of all breast carcinomas, affecting younger women more often and has a worse prognosis than other types of breast cancer, due to the combination of more aggressive clinical behavior and lack of molecular targets for therapy. This study assessed the effects of non-genotoxic concentrations of tributyltin isothiocyanate (TBT-ITC) and triphenyltin isothiocyanate (TPT-ITC) on MDA-MB-231 cells. MATERIALS AND METHODS: MTT assay, comet assay, kinetic imaging and flow cytometry were used for analysis of MDA-MB-231 cells. RESULTS: The results showed that 100 nM concentration of TBT-ITC and TPT-ITC, that did not affect viability or DNA integrity, slowed-down migration by CD44 down-regulation. Moreover, both compounds demonstrated immunomodulatory properties, attenuating PD-L1 expression in MDA-MB-231 cells. CONCLUSION: TPT-ITC was more effective in down-regulating CD44 expression and reducing migration than TBT-ITC, while TBT-ITC was more potent in lowering PD-L1 expression in comparison with TPT-ITC.

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.

Comparative study of relationship between structure of phenylethanoid glycopyranosides and their activities using cell-free assays and human cells cultured in vitro.

The study focused on protective potential of phytochemicals applicable in prevention and health protection is of great importance. Various structures of these compounds and a wide range of their biological activities have inspired organic chemists to sythesize their effective analogues in order to further increase their efficacy. The aims of our study were (i) to synthesize phenylethanoid glycopyranosides: salidroside (SALI - tyrosol ß-d-glucopyranoside), tyrosol ß-d-galactopyranoside (TYBGAL), tyrosol α-d-galactopyranoside (TYAGAL), tyrosol α-d-mannopyranoside (TYAMAN), hydroxytyrosol α-d-mannopyranoside (HOTAMA), homosyringyl ß-d-glucopyranoside (HSYGLU), hydroxytyrosol ß-d-xylopyranoside (HOTXYL) and hydroxysalidroside (HOSALI); (ii) to determine their antioxidant capacities (cell-free approaches); (iii) to evaluate their cytotoxicity (MTT test), protectivity against hydrogen peroxide (H2O2; comet assay) and effect on the intracellular glutathione level (iGSH; flow cytometry) in experimental system utilizing human hepatoma HepG2 cells. HOSALI, HOTAMA, HOTXYL and HSYGLU manifested the highest antioxidant capacity in cell-free assays and they were most active in protection of HepG2 cells against H2O2. On the other hand, pre-treatment of HepG2 cells with SALI had protective effects even though SALI displayed almost no activity in cell-free assays. Differences in the efficacy of the analogues revealed that structures of their molecules in terms of aglycone combined with sugar moiety affect their activities.

Salidroside, a Chemopreventive Glycoside, Diminishes Cytotoxic Effect of Cisplatin in Vitro.

Natural products represent the source or the inspiration for the majority of the active ingredients of medicines because of their structural diversity and a wide range of biological effects. Our aims in this study were (i) to synthesize enzymatically salidroside (SAL), the most effective phenylethanoid glycoside in Rhodiola species; (ii) to examine its antioxidant capacity using cell-free assays (reducing power, DPPH radicals scavenging and Fe2+ -chelating assays); (iii) to assess its DNA-protective potential on plasmid DNA (DNA topology assay) and in HepG2 cells (comet assay) damaged by Fe2+ ions and hydrogen peroxide, respectively; and (iv) to investigate the effects of SAL, cisplatin (CDDP) and combined treatments of SAL + CDDP on cell viability (MTT test), level of DNA damage (comet assay), proliferation, cell cycle (flow cytometry) and the expression of signalling molecules associated with cell growth and apoptotic pathways (Western immunoblotting). We found out that SAL manifested low antioxidant and DNA-protective capacity in all assays used. In both parental A2780 and CDDP-resistant A2780/CP human ovarian carcinoma cells, SAL itself exerted in fact no impact on the viability, while in combination with CDDP it showed antagonistic effect supporting the chemopreventive activity on the CDDP-induced cell damage. These results were confirmed by the partial reversal of the cell cycle alterations and the DNA damage level, as well as with partial restoration of cell survival/signalling pathways, when the expression of these molecules partially returned to their proper levels.

Low numbers of pre-leukemic fusion genes are frequently present in umbilical cord blood without affecting DNA damage response.

Despite widely accepted notion that many childhood leukemias are likely developed from hematopoietic stem/progenitor cells (HSPC) with pre-leukemic fusion genes (PFG) formed in embryonic/fetal development, the data on PFG incidence in newborns are contradictive. To provide a better understanding of a prenatal origin of leukemia, umbilical cord blood from 500 newborns was screened for the presence of the most frequent PFG associated with pediatric B-cell acute lymphoblastic leukemia. This screening revealed relatively high incidence of ETV6-RUNX1, BCR-ABL1 (p190) and MLL-AF4 at very low frequencies, averaging ~14 copies per 100,000 cells. We assume that most of these PFG might originate relatively late in embryonic/fetal development and will be eliminated later during postnatal development. The obtained results suggested that higher PFG copy numbers originating in specific time windows of the hematopoietic stem cell hierarchy may define a better prognostic tool for the assessment of leukemogenic potential. We have observed no significant effect of low-copy PFG on radiation-induced DNA damage response, accumulation of endogenous DNA double-stranded breaks, and apoptosis in either lymphocytes or HSPC. Imaging flow cytometry showed lower level of γH2AX foci in HSPC in comparison to lymphocytes suggesting better protection of HSPC from DNA damage.

Nature of DNA lesions induced in human hepatoma cells, human colonic cells and human embryonic lung fibroblasts by the antiretroviral drug 3'-azido-3'-deoxythymidine.

This study tried to clarify the question if nuclear genotoxicity played a role in 3'-azido-3'-deoxythymidine (AZT) toxicity. We investigated cytotoxic and DNA-damaging effects of AZT on human hepatoma HepG2 and human colonic CaCo-2 cells as well as on human diploid lung fibroblasts HEL. The amount of induced DNA damage was measured by standard alkaline single cell gel electrophoresis (SCGE). The nature of induced DNA lesions was evaluated (1) by modified SCGE, which includes treatment of lysed cells with DNA repair enzymes Endo III and Fpg and enables to recognize oxidized bases of DNA, and (2) by SCGE processed in parallel at pH 13.0 (standard technique) and pH 12.1, which enables to recognize alkali labile DNA lesions and direct DNA strand breaks. Cytotoxicity of AZT was evaluated by the trypan blue exclusion technique. Our findings showed that 3-h treatment of cells with AZT decreased the viability of all cell lines studied. SCGE performed in the presence of DNA repair enzymes proved that AZT induced oxidative lesions to DNA in all cell types. In hepatoma HepG2 cells and embryonic lung fibroblasts HEL the majority of AZT-induced DNA strand breaks were pH-independent, i.e. they were identified at both pH values (12.1 and 13.0). These DNA lesions represented direct DNA breaks. In colonic Caco-2 cells DNA lesions were converted to DNA strand breaks particularly under strong alkaline conditions (pH>13.0), which is characteristic for alkali-labile sites of DNA. DNA strand break rejoining was investigated by the standard comet assay technique during 48 h of post-AZT-treatment in HepG2 and Caco-2 cells. The kinetics of DNA rejoining, considered an indicator of DNA repair, revealed that AZT-induced DNA breaks were repaired in both cell types slowly, though HepG2 cells seemed to be more repair proficient with respect to AZT-induced DNA lesions.

Study of cytotoxic, genotoxic and DNA-protective effects of selected plant essential oils on human cells cultured in vitro.

OBJECTIVES: To investigate cytotoxic, genotoxic and DNA-protective effects of carvacrol and thymol on human hepatoma HepG2 and colonic Caco-2 cells cultured in vitro. METHODS AND RESULTS: Cytotoxicity testing was performed by the trypan blue exclusion technique. Level of DNA lesions induced in human cells with carvacrol, thymol or their combinations with hydrogen peroxide (H(2)O(2)) were measured by alkaline single cell gel electrophoresis (comet assay). The trypan blue exclusion technique showed that though the metabolically more competent human hepatoma HepG2 cells were more sensitive to the toxic effects of carvacrol or thymol than colonic Caco-2 cells, which contained lower levels of metabolizing enzymes, the observed differences were not very expressive. The comet assay technique showed that at concentrations

Rosemary-stimulated reduction of DNA strand breaks and FPG-sensitive sites in mammalian cells treated with H2O2 or visible light-excited Methylene Blue.

In this study possible protective effects of rosemary against oxidative DNA damage induced by H2O2- and visible light-excited Methylene Blue in colon cancer cells CaCo-2 and hamster lung cells V79 were investigated. The level of DNA damage (DNA strand breaks) was measured using the classical and modified single cell gel electrophoresis, so-called comet assay. Our findings showed that an ethanol extract from rosemary reduced the genotoxic activity of both agents after a long-term (24 h; 0.3 microg/ml) or short-term (2 h; 30 microg/ml) pre-incubation of cells. We suggest that the extract of rosemary exhibits a protective effect against oxidative damage to DNA as a consequence of scavenging of both *OH radicals and singlet oxygen ((1)O2).

Molecular and cellular influences of butylated hydroxyanisole on Chinese hamster V79 cells treated with N-methyl-N'-nitro-N-nitrosoguanidine: antimutagenicity of butylated hydroxyanisole.

The antioxidant butylated hydroxyanisole (BHA) is a rodent carcinogen that also reduces the mutagenicity and carcinogenicity of other agents. In this study, we have evaluated possible mechanisms for the antimutagenicity of BHA by investigating its effects on N-methyl-N'-nitro-N-nitrosoguanidine (MNNG)-treated Chinese hamster V79 cells. Mutant frequency was determined using the hprt/V79 assay, while plating efficiency was used to measure cytotoxicity, and apoptosis was measured by flow immunofluorocytometry. In addition, DNA strand breaks and the kinetics of strand-break rejoining were investigated by the alkaline elution of DNA and by single-cell gel electrophoresis (SCGE). Although the higher concentration of BHA (0.5 mM) increased the cytotoxicity of MNNG and the lower concentration of BHA (0.25 mM) did not change it, both concentrations were antimutagenic in MNNG-treated cells, with the greater effect occurring at the lower BHA concentration. Neither BHA nor MNNG nor BHA + MNNG increased the level of apoptotic nuclei, and BHA did not change the level of MNNG-induced DNA strand breaks, though it did inhibit their rejoining. Determination of O(6)-methylguanine-DNA-methyltransferase (MGMT) activity confirmed that V79 cells do not synthesize active MGMT protein; MGMT activity was also undetectable after MNNG and BHA + MNNG treatment. The ability of BHA to reduce the level of MNNG-induced mutations did not correlate with cytotoxicity, induction of apoptosis, the level of DNA strand break induction, or MGMT activity. A modified SCGE assay showed that BHA significantly reduced the level of formamidopyrimidine-DNA-glycosylase + endonucleaseIII-sensitive sites, which at least partially are caused by oxidative DNA lesions. The results suggest that the protective effect of BHA on MNNG-induced mutagenicity is best explained by the antioxidative activity of BHA, which may scavenge free radicals that participate in MNNG-induced mutagenicity.