DNA ligand Hoechst-33342 enhances UV induced cytotoxicity in human glioma cell lines

The effects of minor groove binding ligand bisbenzimidazole derivative Hoechst-33342 on the cellular response to UV damage have been studied in two human glioma cell lines BMG-1 and U-87 grown as monolayer cultures. Treatment induced cell death (macro-colony assay) and growth inhibition, potential lethal damage recovery, cytogenetic damage (micronuclei formation) and proliferation kinetics were studied as parameters for cellular response. Pre and post-irradiation treatment with Hoechst-33342 (1-20 microM) enhanced the UV-induced growth inhibition and cell death in a concentration dependent manner in both cell lines. At higher Hoechst-33342 concentrations (>5 microM), the cytotoxic effects of the combination (Hoechst-33342+UV) were highly synergistic and mainly mediated through apoptosis implying the possible interactions of lesions caused by both the agents. The enhanced cell death due to Hoechst-33342 was accompanied by a significant increase (2-3 folds at 5 microM) in UV-induced micronuclei formation in BMG-1 cells. Under these conditions, Hoechst-33342 also enhanced the UV-induced cell cycle delay, mainly due to S and G(2) blocks. The increase in UV-induced micronuclei formation observed after treatment with Hoechst-33342 indicates that the DNA bound Hoechst-33342 may interfere with the rejoining of DNA strand breaks. Since the treatment of cells with the replication inhibitor aphidicolin reduced the enhancement of UV induced cytotoxicity by Hoechst-33342, ongoing DNA replication appears to stimulate Hoechst-33342 and UV-induced cytotoxicity.     

SPME determination of volatile aldehydes for evaluation of in-vitro antioxidant activity

The in-vitro antioxidant activity of natural (essential oils, vitamin E) or synthetic substances ( tert-butyl hydroxy anisole (BHA), Trolox) has been evaluated by monitoring volatile carbonyl compounds released in model lipid systems subjected to peroxidation. The procedure employed methodology previously developed for the determination of carbonyl compounds as their pentafluorophenylhydrazine derivatives which were quantified, with high sensitivity, by means of capillary gas chromatography with electron-capture detection. Linoleic acid and sunflower oil were used as model lipid systems. Lipid peroxidation was induced in linoleic acid by the Fe2+ ion (1 mmol L-1, 37 degrees C, 12 h) and in sunflower oil by heating in the presence of O2 (220 degrees C, 2 h). The change in hexanal (the main lipoxidation product) concentration found in the lipid matrix subjected to oxidation with and without the substance being tested was used to calculate the antioxidant protection effect. These procedures were employed to evaluate the antioxidant activity of the essential oils of cilantro ( Coriander sativum L.), fennel ( Foeniculum vulgare Mill.), rosemary ( Rosmarinus officinalis L.), "salvia negra" ( Lepechinia schiedeana), and oregano ( Origanum vulgare L.), and the well-known antioxidants BHA, vitamin E, and Trolox, its water-soluble analog. In the sunflower oil system, the essential oils had a stronger protective effect against lipid peroxidation than BHA, vitamin E, and Trolox within the range of concentrations examined (1-20 g L-1). The highest protecting effect, corresponding to a 90% drop in hexanal release, was observed for cilantro oil at 10 g L-1.     

Effects of 50 Hz sinusoidal magnetic fields and spark discharges on human lymphocytes in vitro

Human peripheral blood lymphocytes were exposed in vitro to 50 Hz sinusoidal magnetic fields of 30 μT, 300 μT, and 1 mT, and were monitored for genotoxic effects. No effects on chromosomal aberrations, micronuclei or proliferation indices were noticed. A weak effect on sister chromatid exchanges, noted in one series of experiments, could not be verified when the series was repeated. Exposure of whole blood to spark discharges of up to 3.65 kVcm⁻¹ and 2.6 μs duration (10 pulses) did not result In chromosomal aberrations. Cell destruction from spark discharges was extensive. The conductivity of blood during a spark discharge pulse was examined and was shown to decrease by a factor of 2–3 at the beginning of the pulse owing to the β-relaxation of cells. After 0.5 μs the conductivity approached a constant level.     

Joint effects of elevated levels of ultraviolet-B radiation, carbon dioxide and ozone on plants

There is growing interest regarding the joint effects of elevated levels of surface ultraviolet B (UV-B) radiation, carbon dioxide (CO2) and ozone (O3) on plants. Our current knowledge of this subject is too limited to draw any specific conclusions, although one might state that such effects are likely to be highly species dependent and may be more than additive, additive or less than additive. There are a number of uncertainties associated with the experimental protocols used and the conclusions reached in many studies. Nevertheless, in North America, there appear to be genotypes of three monocot crop species (Avena sativa L., Oryza sativa L. and Sorghum vulgare L.); six dicot crops (Cucumis sativus L., Lactuca sativa L., Lycopersicon esculentum Mill., Phaseolus vulgaris L., Pisum sativum L. and Solanum tuberosum L.) and two conifer species (Pinus ponderosa Dougl. and Pinus taeda L.) that may be considered sensitive to the joint effects of elevated levels of UV-B, CO2 and O3. However, to provide a more reliable assessment or validation of the predictions, future research must consider the concept of plant response surfaces and describe them more fully in numerical terms. Achieving that objective will require close cooperation among a number of scientists representing geographic locations with known spatial and temporal differences in UV-B, CO2 and O3 to conduct experiments under their site-specific conditions, using common plant materials and experimental protocols.     

Clastogenic plasma factors in psoriasis--comparison of phototherapy and anti-TNF-α treatments

As previously described, Psoralen plus UVA (PUVA) therapy induces chromosome damage in psoriatic patients. This study evaluates whether these effects are transitory or persistent. In addition, we studied these effects after narrowband UVB (nUVB) and anti-tumor necrosis factor (TNF)-α treatments. Among 40 responder patients, 10 received PUVA, 10 nUVB, 10 Infliximab and 10 Etanercept. Disease activity was determined with Psoriasis Area and Severity Index. Chromosomal breakage was evaluated by the clastogenic factor (CF) test. Potential clastogenic agents, malondialdehyde (MDA) and TNF-α were measured. Before treatment, the plasma-adjusted clastogenic scores (ACS) of patients were increased. During treatment, a further increase in ACS was observed in both phototherapy groups. Chromosome damage persisted for PUVA patients at week 32, while it diminished after nUVB to ACS values lower than before treatment. MDA and TNF-α values were also increased at baseline. MDA decreased during treatment in all groups, but without reaching normal levels. Plasma TNF-α remained unchanged in PUVA and nUVB but decreased in both anti-TNF-α treatment groups. Psoriasis is accompanied by CF-induced chromosomal breakage that increases during PUVA and nUVB treatments. Plasma clastogenic activity persisted in the follow-up after PUVA, while after nUVB ACS returned to values even lower than baseline. Clastogenic activity during the induction phase with anti-TNF-α remained unchanged.     

Evidence for genotoxic effects of resonant ELF magnetic fields

The possibility that extremely low frequency (ELF) magnetic fields affect the genomic integrity of the cell is the objective of this study. Human peripheral lymphocytes (HPLs) were exposed to different exposure conditions combining ac and static magnetic fields. We used the micronuclei (MN) cytogenetic technique, because MN formation is considered as a marker of chromosomal damage produced by genotoxic agents.The first set of experiments were performed at 50 Hz, 150 μT rms and 32 Hz, 75 μT and 150 μT rms magnetic fields with the static geomagnetic field components nulled. No effects were detected using the MN test on HPL as an indicator for cellular genomic damage when the static magnetic field was nulled. Moreover, such exposure to an ac magnetic field does not appear to interfere with the action of a powerful genotoxic agent mytomicin-C (MMC), i.e. there was no synergistic effect.The second set of experiments were conducted exposing cells to 32 Hz, 150 μT and 75 μT rms, parallel to a 42 μT dc magnetic field. The 75 μT rms, 32 Hz exposure condition was chosen to maximize the resonance effect on Ca²⁺ according to parametric resonance theory. We found a statistically significant increase of MN for both exposure conditions. This experiment provides evidence for the genotoxic effects of resonant ELF magnetic fields in human lymphocytes.     

Aggregation of a peptide antibiotic alamethicin at the air-water interface and its influence on the viscoelasticity of phospholipid monolayers

The aggregation properties of an antibiotic membrane-active peptide alamethicin at the air-water interface have been studied using interfacial rheology and fluorescence microscopy techniques. Fluorescence microscopy of alamethicin monolayers revealed a coexistence of liquid expanded (LE) and solid phases at the surface concentrations studied. Interfacial oscillatory shear measurements on alamethicin monolayers indicate that its viscoelastic properties are determined by the area fraction of the solid domains. The role of zwitterionic phospholipids dioleoylphosphatidyl choline (DOPC) and dioleoylphosphatidyl ethanolamine (DOPE) on the peptide aggregation behavior was also investigated. Fluorescence microscopy of alamethicin/phospholipid monolayers revealed an intermediate phase (I) in addition to the solid and LE phase. In mixed monolayers of phospholipid (L)/alamethicin (P), with increase in L/P, the monolayer transforms from a viscoelastic to a viscous fluid with the increase in area fraction of the intermediate phase. Further, a homogeneous mixing of alamethicin/lipid molecules is observed at L/P > 4. Our studies also confirm that the viscoelasticity of alamethicin/phospholipid monolayers is closely related to the alamethicin/phospholipid interactions at the air-water interface.     

Genotoxic effects of cadmium chloride on human amniotic fluid cells cultured in vitro

In this study we report the results of cytogenetic tests, namely a search for chromosome aberrations (CA) and sister chromatid exchanges (SCEs), performed on human amniotic fluid cells cultured and treated with Cadmium chloride. The cells from primary cultures were exposed to CdCl2 at 1 microM and 10 microM for 24 h. At the higher dose, no metaphases were scored and at the lower dose (1 microM) no effects were evident on cell proliferation, and no chromosome aberrations were found. In the subsequent experiments we used cells from subcultures exposed to 1 microM and 5 microM CdCl2. At the 5 microM dose was evident the induction of chromatid breaks, while the frequency of sister chromatid exchanges shows a small increase, not statistically significant at the dose of 1 microM. In this study we positively demonstrated that amniotic fluid cells grown in vitro are reliable for testing various mutagenic or teratogenic substances. With regard to cadmium treatment results, it is evident a clastogenic effect of cadmium chloride but not a significant induction of SCEs.     

Mitogenic potential inducible by He:Ne laser in human lymphocytes in vitro

The objective of the study was to investigate the mitogenic and genotoxic effects of He:Ne laser irradiation (632.8 nm) on human peripheral lymphocytes in vitro. We used the cytokinesis-block micronucleus assay, which incorporates cytochalasin B to inhibit cytokinesis while karyokinesis proceeds normally leading to the appearance of proliferating lymphocytes as binucleated cells. Also micronuclei will appear in cases of genotoxicologically-affected cells. Buffy coat leukocytes were exposed to 10 mW He:Ne laser at energy densities of 1, 2, 3 and 5 J/cm(2). Cells were then cultured in media 199 without any supplementation for 24, 48, 72 and 96 h adding cytochalasin B 24 h before harvesting of cells. Our results showed that laser-induced lymphocytes proliferate throughout the four consecutive days post-laser irradiation. The difference in the frequency of micronuclei between pre- and post-laser irradiation indicates that a He:Ne laser at such energy densities 1, 2, 3 and 5 J/cm(2) does not induce micronucleus formation. These results shed some light on the mechanism encountered by lymphocytes in the process of He:Ne laser-induced biostimulation.     

The Induction and Analysis of Micronuclei and Cell Killing by Ultraviolet-B Radiation in Human Peripheral Blood Lymphocytes

The DNA-damaging potential of ultraviolet-B (UVB) radiation was investigated by analyzing the frequency and origin of micronuclei (MN) in cytokinesis-blocked, binucleated (BN) peripheral blood lymphocytes (PBL) and cloning efficiencies (CE) of PBL after exposure to different fluences of UVB. In total, PBL obtained from five normal donors were investigated. The PBL from all donors showed a dose-related, linear-quadratic increase in the frequency of MN per 1000 BN cells and in the frequency of micronucleated BN cells. In two experiments the origin of UVB-induced MN was studied by analyzing MN for the presence or absence of centromeres by applying the MN assay in combination with a centromeric probe and fluorescence in situ hybridization. This revealed, for the first time, that UVB-induced MN were centromere negative, indicating that UVB acted exclusively as a clastogenic agent in the tested dose range. The PBL from all donors showed a clear dose-dependent decrease in CE, after UVB exposure. The UVB-exposed PBL from all donors showed an inverse relationship between the induction of MN and the decrease in CE, but regression analysis revealed no correlation between the induction of MN and the decrease in cell survival. It is concluded that UVB has a clastogenic and cytotoxic effect on PBL.     

CYTOTOXICITY AND GENOTOXICITY OF UVA IRRADIATION IN CHINESE HAMSTER OVARY CELLS MEASURED BY SPECIFIC LOCUS MUTATIONS, SISTER CHROMATID EXCHANGES AND CHROMOSOME ABERRATIONS

Abstract— The increasing use of artificial UVA (320-400 nm) suntanning devices has brought attention to possible hazardous effects of UVA. In contrast with earlier studies, several groups recently have described that UVA possibly is mutagenic. In this paper we evaluate the genotoxic properties of broad band UVA using CHO cells and three different assays: specific locus (HGPRT) mutations, chromosome aberrations, and sister chromatid exchanges (SCEs). The UVA-source was an UVASUN 2000 S (Mutzhas), emitting UVA above 340 nm. The survival curve of the cells exhibited a shoulder up to 200 kJ/m², that was followed by exponential killing at higher fluences. Mutations were induced linearly in the fluence range from 0-200 kJ/m² ( P < 0.001) to a level seven fold higher than the spontaneous, followed by a decrease at fluences above 300 kJ/m². Over the total range of tested fluences (0-300 kJ/m²) a linear dose-response relationship was observed for UVA-induced SCEs ( P < 0.001). A significantly higher percentage of the cells showed chromosomes with aberrations at the higher levels of exposure (200, 300 and 400 kJ/m²), but no dose response was demonstrated. Our results confirm recent findings showing that UVA is mutagenic in mammalian cells and suggest that UVA exposure may contribute to the total burden of genetic damage caused by exposure to ultraviolet light.     

Antioxidant capacity of Ocimum basilicum L. and Origanum vulgare L. extracts

The antioxidant properties of five different extracts (Et?O, CHCl?, EtOAc, n-BuOH, and H?O) of Ocimum basilicum L. and Origanum vulgare L. were studied. Antioxidant activity was assessed in six different model systems. Free radical scavenging capacity (RSC) was evaluated by measuring the scavenging capacity of extracts on DPPH, NO, O??? and OH radical, as well as on hydrogen peroxide (H?O?). In addition, the protective effects on lipid peroxidation in liposomes (LPx) were evaluated by TBA-assay using the Fe2?/ascorbate induction system. The amount of total phenolic compounds and content of total flavonoids was also determined. EtOAc, n-BuOH and H?O extracts of O. basilicum and O. vulgare expressed very strong scavenger activity. Furthermore, the mentioned extracts showed notable inhibition of LPx. On the other hand, Et?O and CHCl? extracts showed much weaker effect in the neutralization of DPPH, NO and O??? radicals and the neutralization of H?O?. When examining the production of OH radicals and inhibition of LPx, the Et?O and CHCl? extracts showed weak prooxidative properties. The observed differences in antioxidant activity could be partially explained by the levels of phenolics and flavonoids in the investigated O. basilicum and O. vulgare extracts.     

Extraction of volatile oil from aromatic plants with supercritical carbon dioxide: experiments and modeling

An overview of the studies carried out in our laboratories on supercritical fluid extraction (SFE) of volatile oils from seven aromatic plants: pennyroyal (Mentha pulegium L.), fennel seeds (Foeniculum vulgare Mill.), coriander (Coriandrum sativum L.), savory (Satureja fruticosa Béguinot), winter savory (Satureja montana L.), cotton lavender (Santolina chamaecyparisus) and thyme (Thymus vulgaris), is presented. A flow apparatus with a 1 L extractor and two 0.27 L separators was built to perform studies at temperatures ranging from 298 to 353 K and pressures up to 30.0 MPa. The best compromise between yield and composition compared with hydrodistillation (HD) was achieved selecting the optimum experimental conditions of extraction and fractionation. The major differences between HD and SFE oils is the presence of a small percentage of cuticular waxes and the relative amount of thymoquinone, an oxygenated monoterpene with important biological properties, which is present in the oils from thyme and winter savory. On the other hand, the modeling of our data on supercritical extraction of volatile oil from pennyroyal is discussed using Sovová's models. These models have been applied successfully to the other volatile oil extractions. Furthermore, other experimental studies involving supercritical CO₂ carried out in our laboratories are also mentioned.     

Chromium (VI) ion adsorption features of chitosan film and its chitosan/zeolite conjugate 13X film

This research evaluated the importance of the adsorption properties of chitosan a chitosan/zeolite conjugate film for the removal of Cr(VI) ions from solutions in the 5-260 mg/L concentration range, when the pH was adjusted to 4.0 and 6.0. The uptake capacities of the films formed by chitosan and by the chitosan/zeolite conjugate were calculated by mass balance. The equilibrium isotherms were fitted to the Langmuir, Freundlich and Redlich-Peterson models. The chitosan film seems to be a good sorbent for Cr(VI) at pH 4, but its physical instability suggests the need for a more resilient support. Due to this fact zeolite was added to the chitosan matrix in solution and a chitosan/zeolite (CS/Zeo) film was thus formed. The solubility of the film and the characterization of the different matrices by FTIR, TGA and X-Ray showed that a cross-linked structure was formed between the chitosan and zeolite and the solubility of the film increased. In this study, the low manufacturing cost of the CS/Zeo matrix, the good uptake of Cr(VI) at acidic pH (17.28 mg/g) and the non desorption of Cr(VI) from the film in water suggests this combination should be tested in industrial environment.     

Analysis of the essential oils of Coriandrum sativum Using GC-MS coupled with chemometric resolution methods

The essential oils extracted from Coriandrum sativum L. were analyzed by GC-MS coupled with chemometric resolution methods. Through the chemometric resolution methods, peak clusters were uniquely resolved into the pure chromatographic profiles and mass spectra of each component. Qualitative analysis was performed by comparing the pure mass spectra with those in the NIST 05 mass spectral library. Quantitative analysis was performed using the total volume integration method. A total of 118 constituents were detected, of which 104 were identified, accounting for 97.27% of the total content. The results indicate that GC-MS combined with chemometric resolution methods can greatly enhance the capability of separation and the reliability of qualitative and quantitative results. The combined method is an economical and accurate approach for the rapid analysis of the complex essential oil samples in Coriandrum sativum L.     

In vitro inhibitory activity of essential oil vapors against Ascosphaera apis

This work evaluates the in vitro inhibitory activity of 70 essential oils (EOs) in the vapor phase for the control of Chalkbrood disease caused by Ascosphaera apis Maassen ex Claussen (Olive et Spiltoir). Two wild strains isolated from infected honey bee colonies together with one standard collection strain were tested by the microatmosphere method. From 70 EOs, 39 exhibited an antifungal effect against A. apis standard and wild strains. The greatest antifungal action was observed for EO vapors from Armoracia rusticana, followed by Thymus vulgaris, Cymbopogon flexosus, Origanum vulgare and Allium sativum. An investigation of chemical composition by GC-MS revealed, that the most active EOs contained allyl isothiocyanate, citral, carvacrol and diallyl sulfides as the main constituents. The chemical composition plays a key role, as activities of different EOs from the same botanical species were different according to their composition.     

解偶联剂CCCP对莱茵衣藻光照产氢过程的调控

本文研究莱茵衣藻在含硫和缺硫连续光照条件下, 不同浓度的CCCP对PSⅡ光化学效率、 光照产氢、 光合放氧以及光能的吸收和转换效率等的影响和调控规律.     

Genotoxicity of visible light (400-800 nm) and photoprotection assessment of ectoin, L-ergothioneine and mannitol and four sunscreens

This study was designed to determine the genotoxic effects of visible (400-800nm) and ultraviolet A (UVA)/visible (315-800nm) lights on human keratinocytes and CHO cells. The alkaline comet assay was used to quantify DNA-damage. In addition, photo-dependent cytogenetic lesions were assessed in CHO cells by the micronucleus test. Three protective compounds [ectoin, l-ergothioneine (ERT) and mannitol] were tested with the comet assay for their effectiveness to reduce DNA single-strand breaks (SSB). Finally, the genomic photoprotections of two broad-band sunscreens and their tinted analogues were assessed by the comet assay. The WST-1 cytotoxicity assay revealed a decrease of the keratinocyte viability of 30% and 13% for the highest UVA/visible and visible irradiations (15 and 13.8J/cm(2), respectively). Visible as well as UVA/visible lights induced DNA SSB and micronuclei, in a dose-dependent manner. The level of DNA breakage induced by visible light was 50% of the one generated by UVA/visible irradiation. However, UVA radiations were 10 times more effective than visible radiations to produce SSB. The DNA lesions induced by visible and UVA/visible lights were reduced after a 1-h preincubation period with the three tested compounds. The maximal protective effects were 92.7%, 97.9% and 52.0% for ectoin (0.1mM), ERT (0.5mM) and mannitol (1.5mM), respectively, against visible light and 68.9%, 59.8% and 62.7% for ectoin (0.1mM), ERT (0.5mM) and mannitol (1.5mM), respectively, against UVA/visible light. Thus, visible light was genotoxic on human keratinocytes and CHO cells through oxidative stress mechanisms similar to the ones induced by UVA radiations. The four tested sunscreens efficiently prevented DNA lesions that were induced by both visible and UVA/visible irradiations. The tinted sunscreens were slightly more effective that their colorless analogues. There is a need to complement sunscreen formulations with additional molecules to obtain a complete internal and external photoprotection against both UVA and visible lights.     

Distribution of coexisting solid and fluid phases alters the kinetics of collapse from phospholipid monolayers

To determine how coexistence of liquid-expanded (LE) and tilted-condensed (TC) phases in phospholipid monolayers affects collapse from the air/water interface, we studied binary films containing dioleoyl phosphatidylcholine-dipalmitoyl phosphatidylcholine (DPPC) mixtures between 10 and 100% DPPC. Previously published results established that this range of compositions represents the LE-TC coexistence region at the equilibrium spreading pressure of 47 mN/m. When held at 49.5 mN/m on a captive bubble, the extent of total collapse fit with the LE area predicted by the phase diagram. The kinetics of collapse, however, when normalized for changes in the LE area, slowed with increasing mole fraction of DPPC. Surface area expressed as stretched exponential functions of time yielded an Avrami exponent that decreased from 1 for the homogeneously LE film to 0.3 for DPPC > or = 70%. Microscopic studies showed that the largest changes in kinetics occurred when either alterations of the initial composition or the process of collapse induced the films to cross the percolation threshold, so that the LE phase became divided into isolated domains. Our results show that although coexisting solid and fluid phases collapse to extents that are independent, the kinetics of collapse, corrected for differences in LE area, depend on the distribution of the two phases.     

Medicinal foodstuffs. XXV. Hepatoprotective principle and structures of ionone glucoside, phenethyl glycoside, and flavonol oligoglycosides from young seedpods of garden peas, Pisum sativum L

A new ionone glucoside, pisumionoside, a phenethyl glycoside, sayaendoside, and two acylated flavonol oligoglycosides, pisumflavonosides I and II, were isolated from the young seedpods of garden peas, Pisum sativum L., together with quercetin and kaempferol 3-O-(6-O-trans-p-coumaroyl)-beta-D-glucopyranosyl (1-->2)-beta-D-glucopyranosyl (1-->2)-beta-D-glucopyranosides and quercetin and kaempferol 3-sophorotriosides. The structures of pisumionoside, sayaendoside, and pisumflavonosides I and II were determined on the basis of chemical and physicochemical evidence, respectively. Quercetin 3-sophorotrioside, a principle component, was found to show protective effects on liver injury induced by D-galactosamine and lipopolysaccharide and by carbon tetrachloride in mice.