Genotoxic activity of 1,2,3-triazolyl modified furocoumarins and 2,3-dihydrofurocoumarins.

The furocoumarin backbone is a promising platform for chemical modifications aimed at creating new pharmaceutical agents. However, the high level of biological activity of furocoumarins is associated with a number of negative effects. For example, some of the naturally occurring ones and their derivatives can show genotoxic and mutagenic properties as a result of their forming crosslinks with DNA molecules. Therefore, a particularly important area for the chemical modification of natural furocoumarins is to reduce the negative aspects of their bioactivity. By studying a group of 21 compounds-1,2,3-triazolyl modified derivatives of furocoumarin and peucedanin-using the SOS chromotest, the Ames test, and DNA-comet assays, we revealed modifications that can neutralize the structure's genotoxic properties. Theoretical aspects of the interaction of the compound library were studied using molecular modeling and this identified the leading role of the polyaromatic molecular core that takes part in stacking-interactions with the pi-systems of the nitrogenous bases of DNA.

In vitro and in vivo toxicity assessment of alginate/eudragit S 100-enclosed chitosan-calcium phosphate-loaded iron saturated bovine lactoferrin nanocapsules (Fe-bLf NCs).

Lactoferrin has been known to have antimicrobial properties. This research was conducted to investigate the toxicity of Alginate/EUDRAGIT® S 100-enclosed chitosan-calcium phosphate-loaded Fe-bLf nanocapsules (NCs) by in vitro and in vivo assays. Brine shrimp lethality assay showed that the LC50 value of NCs was more than 1mg/mL which indicated that NCs was not toxic to Brine shrimp. However, the LC50 values for the positive control potassium dichromate at 24h is 64.15µg/mL, which was demostrated the toxic effect against the brine shrimp. MTT cytotoxicity assay also revealed that NCs was not toxic against non-cancerous Vero cell line with IC50 values of 536µg/mL. Genotoxicity studies by comet assay on Vero cells revealed that NCs exerted no significant genotoxic at 100µg/mL without tail or shorter comet tail. Allium cepa root assay carried out at 125, 250, 500 and 1000µg/mL for 24h revealed that the NCs was destitute of significant genotoxic effect under experimental conditions. The results show that there is no significant difference (p>0.05) in mitotic index between the deionized water and NCs treated Allium cepa root tip cells. In conclusion, no toxicity was observed in NCs in this study. Therefore, nontoxic NCs has the good potential to develop as a therapeutic agent.

Cytotoxicity testing of aqueous extract of bitter leaf (Vernonia amygdalina Del) and sniper 1000EC (2,3 dichlorovinyl dimethyl phosphate) using the Alium cepa test.

BACKGROUND: The unrefined nature of the herbal preparations from Vernonia amygdalina (VA) and toxicity potentials of Sniper may both have severe consequences on the biochemical and genetic systems. OBJECTIVES: To assess the microscopic and macroscopic effects of these substances. METHODS: VA leafs and Sniper were prepared and dissolved in distilled water to give different concentrations. Series of baseline tests were carried out to establish concentration range for root growth. Series of twelve onion bulbs of three per series was prepared, with a series of three onion bulbs serving as control. Chromosomal aberrations were statistically analysed using chi- squared test. Root bundle mean length was obtained after 96 hours and EC50 values at 95% confidence interval was determined from a plot of root length against sample concentrations using Microsoft Excel software. RESULTS: Total cytotoxic effect was induced by 2% sniper and 70% VA. EC50 for VA and sniper were 33.07 and 0.346 respectively. The two substances induced chromosomal aberrations and the effect was concentration dependent. CONCLUSION: There are risks of these widely used substances for therapeutic and environmental purposes.

New antimony(III) halide complexes with dithiocarbamate ligands derived from thiuram degradation: The effect of the molecule's close contacts on in vitro cytotoxic activity.

Antimony(III) halide complexes of the formulae {[SbBr(Me2DTC)2]n} (1), {[SbI(Me2DTC)2]n} (2) and {[(Me2DTC)2Sb(µ2-I)Sb(Me2DTC)2](+).I3(-)} (3) (Me2DTC = dimethyldithiocarbomate) were synthesized from SbX3, (X = Br or I) and tetramethylthiuram monosulfide (Me4tms) or tetramethylthiuram disulfide (Me4tds). The complexes were characterized by melting point (m.p.), elemental analysis (e.a.), Fourier-transform Infra-Red (FT-IR), Fourier-transform Raman (FT-Raman), Nuclear Magnetic Resonance ((1)H,(13)C-NMR) spectroscopy and Thermogravimetric-Differential Thermal Analysis (TG-DTA). Crystal structures of complexes 1-3 were determined with single crystal X-ray diffraction analysis. Complexes 1 and 2 are polymers with distorted square pyramidal (SP) geometry in each monomeric unit, whereas complex 3 is ionic, containing an iodonium linkage Sb-I(+)-Sb and an I3(-) counter anion; to the best of our knowledge, this is the first ionic antimony(III) iodide complex. The in vitro cytotoxic activity of 1-3 against human adenocarcinoma cells: breast (MCF-7) and cervix (HeLa) cells and non-cancerous cells: MRC-5 (normal human fetal lung fibroblast cells) was evaluated with trypan blue (TB) and sulforhodamine B (SRB) assays. Among antimony(III) compounds with sulfur containing ligand, those of dithiocarbamates exhibit significant cytotoxic activity. Hirshfeld surface volumes were analyzed to clarify the nature of the intermolecular interactions by the 2D fingerprint plot. Molecules with lower H-all atoms inter-molecular interactions exhibit the higher activity against MCF-7 cells. The in vivo genotoxicity of 1-3 was evaluated by the mean of Allium cepa test. Alterations in the mitotic index values due to the chromosomal aberrations were observed in the case of complexes 2 and 3. Since, no such alteration is caused by 1, it makes this compound candidate for further study as potential drug.

Intra- and intertissular cytomictic interactions in mic-rosporogenesis of mono- and dicotyledonous plants.

A comparative cytological analysis of intra- and intertissular cytomictic interactions in early micro-sporogenesis of mono- and dicotyledonous plants was performed by the example of the two cellular systems - microsporocytes and tapetum. It is found that cytomixis is the component of intratissular interactions mainly. In the tapetum cells cytomixis is notable for structural and temporary taxon specific features. The nuclear migration in microsporocytes is confined mainly to zygotene-pachytene meiotic stages and characterized by a certain synchronism with cytomixis at the tapetum. Intertissular cytomictic interactions (tapetum - microsporocytes) were found in the monocot anthers only. Intertissular interactions are likely to reflect the intensification of competitive relations between the tapetum and microsporocytes for area in the process of anther tissue differentiation. Polyploid tapetum nucleus and syncytia being powerful acceptors are able to compete with microsporocytes and direct the chromatin translocation to their favor. The absence of intertissular interactions in dicots probably reflects a better balance between the processes of differentiation at somatic and generative tissues into microsporangium compared to monocots.

The effects of anti-DNA topoisomerase II drugs, etoposide and ellipticine, are modified in root meristem cells of Allium cepa by MG132, an inhibitor of 26S proteasomes.

DNA topoisomerase II (Topo II), a highly specialized nuclear enzyme, resolves various entanglement problems concerning DNA that arise during chromatin remodeling, transcription, S-phase replication, meiotic recombination, chromosome condensation and segregation during mitosis. The genotoxic effects of two Topo II inhibitors known as potent anti-cancer drugs, etoposide (ETO) and ellipticine (EPC), were assayed in root apical meristem cells of Allium cepa. Despite various types of molecular interactions between these drugs and DNA-Topo II complexes at the chromatin level, which have a profound negative impact on the genome integrity (production of double-strand breaks, chromosomal bridges and constrictions, lagging fragments of chromosomes and their uneven segregation to daughter cell nuclei), most of the elicited changes were apparently similar, regarding both their intensity and time characteristics. No essential changes between ETO- and EPC-treated onion roots were noticed in the frequency of G1-, S-, G2-and M-phase cells, nuclear morphology, chromosome structures, tubulin-microtubule systems, extended distribution of mitosis-specific phosphorylation sites of histone H3, and the induction of apoptosis-like programmed cell death (AL-PCD). However, the important difference between the effects induced by the ETO and EPC concerns their catalytic activities in the presence of MG132 (proteasome inhibitor engaged in Topo II-mediated formation of cleavage complexes) and relates to the time-variable changes in chromosomal aberrations and AL-PCD rates. This result implies that proteasome-dependent mechanisms may contribute to the course of physiological effects generated by DNA lesions under conditions that affect the ability of plant cells to resolve topological problems that associated with the nuclear metabolic activities.

Probing single-cell mechanics with picosecond ultrasonics.

The mechanical properties of cells play a key role in several fundamental biological processes, such as migration, proliferation, differentiation and tissue morphogenesis. The complexity of the inner cell composition and the intricate meshwork formed by transmembrane cell-substrate interactions demands a non-invasive technique to probe cell mechanics and cell adhesion at a subcell scale. In this paper we review the use of laser-generated GHz acoustic waves--a technique called picosecond ultrasonics (PU)--to probe the mechanical properties of single cells. We first describe applications to vegetal cells and biomimetic systems. We show how these systems can be used as simple models to understand more complex animal cells. We then present an opto-acoustic bio-transducer designed for in vivo measurements in physiological conditions. We illustrate the use of this transducer through the simultaneous probing of the density and compressibility of Allium cepa cells. Finally, we demonstrate that this technique can quantify animal-cell adhesion on metallic surfaces by analyzing the acoustic pulses reflected off the cell-metal interface. This innovative approach allows investigating quantitatively cell mechanics without fluorescent labels or mechanical contact to the cell.

Automated cell-by-cell tissue imaging and single-cell analysis for targeted morphologies by laser ablation electrospray ionization mass spectrometry.

Mass spectrometry imaging (MSI) is an emerging technology for the mapping of molecular distributions in tissues. In most of the existing studies, imaging is performed by sampling on a predefined rectangular grid that does not reflect the natural cellular pattern of the tissue. Delivering laser pulses by a sharpened optical fiber in laser ablation electrospray ionization (LAESI) mass spectrometry (MS) has enabled the direct analysis of single cells and subcellular compartments. Cell-by-cell imaging had been demonstrated using LAESI-MS, where individual cells were manually selected to serve as natural pixels for tissue imaging. Here we describe a protocol for a novel cell-by-cell LAESI imaging approach that automates cell recognition and addressing for systematic ablation of individual cells. Cell types with particular morphologies can also be selected for analysis. First, the cells are recognized as objects in a microscope image. The coordinates of their centroids are used by a stage-control program to sequentially position the cells under the optical fiber tip for laser ablation. This approach increases the image acquisition efficiency and stability, and enables the investigation of extended or selected tissue areas. In the LAESI process, the ablation events result in mass spectra that represent the metabolite levels in the ablated cells. Peak intensities of selected ions are used to represent the metabolite distributions in the tissue with single-cell resolution.

Three-dimensional mid-infrared tomographic imaging of endogenous and exogenous molecules in a single intact cell with subcellular resolution.

Microscopy in the mid-infrared spectral range provides detailed chemical information on a sample at moderate spatial resolution and is being used increasingly in the characterization of biological entities as challenging as single cells. However, a conventional cellular 2D imaging measurement is limited in its ability to associate specific compositional information to subcellular structures because of the interference from the complex topography of the sample. Herein we provide a method and protocols that overcome this challenge in which tilt-series infrared tomography is used with a standard benchtop infrared microscope. This approach gives access to the quantitative 3D distribution of molecular components based on the intrinsic contrast provided by the sample. We demonstrate the method by quantifying the distribution of an exogenous metal carbonyl complex throughout the cell and by reporting changes in its coordination sphere in different locations in the cell.

Pressure-volume curves: revisiting the impact of negative turgor during cell collapse by literature review and simulations of cell micromechanics.

The Scholander-Hammel pressure chamber has been used in thousands of papers to measure osmotic pressure, πc , turgor pressure, Pt , and bulk modulus of elasticity, ε, of leaf cells by pressure-volume (PV) curve analysis. PV analysis has been questioned in the past. In this paper we use micromechanical analysis of leaf cells to examine the impact on PV curve analysis of negative turgor in living cells (Pt ). Models predict negative Pt (-0.1 to -1.8 MPa) depending on leaf cell size and shape in agreement with experimental values reported by J. J. Oertli. Modeled PV curves have linear regions even when Pt is quite negative, contrary to the arguments of M.T. Tyree. Negative Pt is totally missed by PV curve analysis and results in large errors in derived πc and Pt but smaller errors in ε. A survey of leaf cell sizes vs habitat (arid, temperate, and rainforest), suggests that the majority of published PV curves result in errors of 0.1-1.8 MPa in derived πc and Pt , whereby the error increases with decreasing cell size. We propose that small cell size in leaves is an ecological adaptation that permits plants to endure negative values of water potential with relatively little water loss.

Removal of landfill leachate toxicity and genotoxicity by two treatment methods.

Leachates from active and closed municipal solid waste landfills can be a major source of contamination to groundwater and surface waters. In the present study the toxic and genotoxic potential of leachate from an old sanitary landfill prior to and following chemical and electrochemical treatments were assessed using Lemna, Allium, and comet tests. Photosynthetic pigments, malondialdehyde (indicator of lipid peroxidation) and antioxidant enzyme activities were evaluated as additional indicators of toxicity in duckweed. Following duckweed exposure to 25 % dilution of landfill leachate, growth rate and photosynthetic pigments content significantly decreased while lipid peroxidation increased despite stimulation of antioxidative defence mechanisms. Diluted leachate induced DNA strand breaks in duckweed cells as evidenced by the comet assay. Regarding the Allium test, untreated leachate caused inhibition of Allium cepa cell division and induction of mitotic and chromosomal aberrations. Although both water treatments completely reduced genotoxicity of leachate, the electrochemical method was found to be more efficient in removing toxic substances present in landfill leachate and thus more suitable for treating such leachates prior to their discharge into the environment. As landfill leachates pose a risk to human health and environment in general due to their (geno)toxicity, the present study demonstrates that the ecotoxicity/genotoxicity assays should be used in leachate risk assessment together with physicochemical analysis.

Evolution of leaf blade anatomy in Allium (Amaryllidaceae) subgenus Amerallium with a focus on the North American species.

PREMISE OF THE STUDY: Reconstructing ancestral states is a useful method to understand the pathway and patterns of character evolution and to test specific hypotheses within a phylogenetic context. Using a phylogenetic hypothesis of the subgenus Amerallium and related subgenera based on molecular data, we reconstructed the evolutionary history of leaf blade anatomical characters and identified those characters that are most congruent with phylogenetic relationships. Furthermore, we used these character histories to investigate the evolution of terete leaves and explore a possible correlation between environment and leaf anatomy in the North American species. METHODS: Sixty-seven North American and Old World species were sampled from all major infrageneric taxa and lineages for transectional leaf anatomy. To provide a phylogenetic framework for interpretation, representatives of Old World Amerallium and related subgenera were added to a published data matrix of North American taxa and ITS, ETS, trnL-F, and rpL32-trnL sequences. KEY RESULTS: Four anatomical characters, namely leaf-blade shape in transection, presence versus absence of palisade mesophyll, distribution and orientation of vascular bundles, and position of laticifer cells, were found to be congruent with phylogenetic relationships and useful diagnostic traits within North American species. Character reconstructions show that terete leaves in North American species evolved from flattened leaves via a possible transition from subterete to terete leaves. Furthermore, terete leaves possess traits that are indicative of possible adaptation to xeric environments. CONCLUSIONS: The findings from this study provide valuable information for understanding the evolution of leaf-blade anatomy in North American Allium species.

The Allium test: injection of test compounds into the bulb.

We developed three methods for injecting substances into the bulb of Allium: (1) direct injection, (2) injection through the open space in the bulb after the removal of the upper half of the bulb, and (3) through a special "tunnel" cut in the bulb. Water (control) and solutions of methotrexate and copper sulfate were injected into bulbs and the dynamics of root growth were measured. We found that 2.2 mM methotrexate and 100 mM copper sulfate inhibit root growth. Injection through the "tunnel" was the most effective method. Cytological analysis showed that methotrexate inhibited mitotic activity completely in the root apical meristem and copper sulfate induced swelling of cells in the root meristem.

Allium fistulosum as a novel system to investigate mechanisms of freezing resistance.

Allium fistulosum was investigated as a novel model system to examine the mechanism of freezing resistance in cold hardy plants. The 250 × 50 × 90 µm average cell size and single epidermal cell layer system allowed direct observation of endoplasmic reticulum (ER), functional group localization during acclimation, freezing and thawing on an individual cell basis in live intact tissues. Cells increased freezing resistance from an LT50 of -11°C (non-acclimated) to -25°C under 2 weeks of cold acclimation. Samples were processed using Fourier transform infrared technology (FTIR) on a synchrotron light source and a focal plane array detector. In addition, confocal fluorescent microscopy combined with a cryostage using ER selective dye of ER-Tracker allowed more detailed examination of membrane responses during freezing. Cold acclimation increased the ER volume per cell, and the freeze-induced cell deformation stopped ER streaming and ER vesiculation subsequently occurred through the breakdown in the ER network. Freeze-induced ER vesicles in cold-acclimated cells were larger and more abundant than those in non-acclimated cells. According to FTIR, the carbohydrate/ester fraction and α-helical/ß-sheet secondary structure localized in the apoplast/plasma membrane region were most visibly increased during cold acclimation. Results suggest the mechanism of cold acclimation and freezing resistance in very hardy cells may be associated with both alterations in the apoplast/plasma membrane region and the ER cryodynamics. Allium fistulosum appears to be a useful system to obtain direct evidence at both intra and extracellular levels during cold acclimation and the freezing process.

[Mitotoxic and genotoxic effect of sediment on the example of the small rivers of the Orenburg region].

The Purpose--an estimation mitotoxic and genotoxic activities of sediments from small Blyava and Kuraganka rivers (Orenburg region) with the use of ana- telophase method. The results have shown the reduction in mitotoxic and genotoxic activities of the sediments over the 2007-2010 period.

Characterization of callase (ß-1,3-D-glucanase) activity during microsporogenesis in the sterile anthers of Allium sativum L. and the fertile anthers of A. atropurpureum.

We examined callase activity in anthers of sterile Allium sativum (garlic) and fertile Allium atropurpureum. In A. sativum, a species that produces sterile pollen and propagates only vegetatively, callase was extracted from the thick walls of A. sativum microspore tetrads exhibited maximum activity at pH 4.8, and the corresponding in vivo values ranged from 4.5 to 5.0. Once microspores were released, in vitro callase activity peaked at three distinct pH values, reflecting the presence of three callase isoforms. One isoform, which was previously identified in the tetrad stage, displayed maximum activity at pH 4.8, and the remaining two isoforms, which were novel, were most active at pH 6.0 and 7.3. The corresponding in vivo values ranged from pH 4.75 to 6.0. In contrast, in A. atropurpureum, a sexually propagating species, three callase isoforms, active at pH 4.8-5.2, 6.1, and 7.3, were identified in samples of microsporangia that had released their microspores. The corresponding in vivo value for this plant was 5.9. The callose wall persists around A. sativum meiotic cells, whereas only one callase isoform, with an optimum activity of pH 4.8, is active in the acidic environment of the microsporangium. However, this isoform is degraded when the pH rises to 6.0 and two other callase isoforms, maximally active at pH 6.0 and 7.3, appear. Thus, factors that alter the pH of the microsporangium may indirectly affect the male gametophyte development by modulating the activity of callase and thereby regulating the degradation of the callose wall.

Cryobanking of Korean allium germplasm collections: results from a 10 year experience.

This paper reviews a 10-year experience in establishing a cryopreserved Allium germplasm collection at the genebank of the National Agrobiodiversity Center, Republic of Korea. A systematic approach to Allium cryopreservation included: 1. revealing the most critical factors that affected regeneration after cryostorage; 2. understanding the mechanisms of cryoprotection by analyzing the thermal behavior of explants and cryoprotectant solutions using DSC and influx/efflux of cryoprotectants using HPLC; 3. assessing genetic stability of regenerants; and 4. revealing the efficiency of cryotherapy. Bulbil primordia, i.e. asexual bulbs formed on unripe inflorescences, proved to be the most suitable material for conservation of bolting varieties due to high post-cryopreservation regrowth and lower microbial infection level, followed by apical shoot apices from single bulbs and cloves. A total of 1,158 accessions of garlic as well as some Allium species have been cryopreserved during 2005-2010 using the droplet-vitrification technique with a mean regeneration percentage of 65.9 percent after cryostorage. These results open the door for large-scale implementation of cryostorage and for simplifying international exchange for clonal Allium germplasm.

Occurrence of differential meiotic associations and additional chromosomes in the embryo-sac mother cells of Allium roylei Stearn.

A small population of complex translocation heterozygote plants of Allium roylei from the Bani region of Jammu Province was studied for meiosis in the female track. This study resulted in identification of two variants, having embryo-sac mother cells (EMCs) with more than 16 chromosomes. EMCs of the remaining plants invariably had diploid (2n = 16) chromosome complement. Female meiosis, in general, was found to be abnormal, with nearly 23% and 11% chromosomes associating as quadrivalents or trivalents at prophase I and at metaphase I, respectively. This was followed by irregular segregation of chromosomes at anaphase I. Amongst the variants; one had 38% EMCs with eight bivalents plus two small sized chromosomes. Their small size, dispensable nature and tendency to affect the pairing behaviour of normal complement are some of the features that latter chromosomes share with the B chromosomes. Seventeen to nineteen chromosomes were observed in 35% EMCs of other variant; the remaining cells had 16 chromosomes. Chromosomal behaviour in both kind of cells (euploid and aneuploid) was more or less similar. Unlike female meiocytes, male meiocytes analysed earlier of this strain always had 16 chromosomes which paired to form extremely complex associations involving 3-16 chromosomes. The most likely cause of this asynchrony with regards to number of chromosomes involved in multivalent formation seems to be interaction of genes controlling chiasma formation with the different physiological conditions of male and female meiocytes.

In situ cell-by-cell imaging and analysis of small cell populations by mass spectrometry.

Molecular imaging by mass spectrometry (MS) is emerging as a tool to determine the distribution of proteins, lipids, and metabolites in tissues. The existing imaging methods, however, mostly rely on predefined rectangular grids for sampling that ignore the natural cellular organization of the tissue. Here we demonstrate that laser ablation electrospray ionization (LAESI) MS can be utilized for in situ cell-by-cell imaging of plant tissues. The cell-by-cell molecular image of the metabolite cyanidin, the ion responsible for purple pigmentation in onion (Allium cepa) epidermal cells, correlated well with the color of cells in the tissue. Chemical imaging using single-cells as voxels reflects the spatial distribution of biochemical differences within a tissue without the distortion stemming from sampling multiple cells within the laser focal spot. Microsampling by laser ablation also has the benefit of enabling the analysis of very small cell populations for biochemical heterogeneity. For example, with a ∼30 µm ablation spot we were able to analyze 3-4 achlorophyllous cells within an oil gland on a sour orange (Citrus aurantium) leaf. To explore cell-to-cell variations within and between tissues, multivariate statistical analysis on LAESI-MS data from epidermal cells of an A. cepa bulb and a C. aurantium leaf and from human buccal epithelial cell populations was performed using the method of orthogonal projections to latent structures discriminant analysis (OPLS-DA). The OPLS-DA analysis of mass spectra, containing over 300 peaks each, provided guidance in identifying a small number of metabolites most responsible for the variance between the cell populations. These metabolites can be viewed as promising candidates for biomarkers that, however, require further verification.

Evaluation of cytotoxicity and genotoxicity of Inula viscosa leaf extracts with Allium test.

I. viscosa has been used for years in folk medicine for its anti-inflammatory, antipyretic, antiseptic, and paper antiphlogistic activities. In this study, cytotoxic and genotoxic effects of I. viscosa leaf extracts on the root meristem cells of Allium cepa have been examined. Onion bulbs were exposed to 2.5 mg/ml, 5 mg/ml, and 10 mg/ml concentrations of the extracts for macroscopic and microscopic analysis. Tap water has been used as a negative control and Ethyl methanesulfonate (EMS) (2 * 10(-2) M) has been used as a positive control. The test concentrations have been determined according to doses which are recommended for use in alternative medicine. There has been statistically significant (P < .05) inhibition of root growth depending on concentration by the extracts when compared with the control groups. All the tested extracts have been observed to have cytotoxic effects on cell division in A. cepa. I. viscosa leaf extract induces the total number of chromosomal aberrations and micronuclei (MNC) formations in A. cepa root tip cells significantly when compared with control groups. Also, this paper shows for the first time the induction of cell death, ghost cells, cells with membrane damage, and binucleated cells by extract treatment. These results suggest the cytotoxic and genotoxic effects of the I. viscosa leaf extracts on A. cepa.