Thrombolysis, clotting, and genotoxic activities modulated by essential oils extracted from Lippia alba

Abstract The composition and pharmacological properties of Lippia alba (Mill.) (L. alba) (Verbenaceae) flower and leaf essential oils (EO) were determined in this study. The major constituents in the flower EO were geranial (49.83%) and neral (32.75%), and in the leaf EO were geranial (38.06%), neral (31.02%), and limonene (18.03%). Flower EO inhibited thrombolysis induced by Bothrops moojeni (B. moojeni) and Lachesis muta muta (L. muta muta) venoms (0.05-1.2 µL mL-1). When tested against L. muta muta venom, the protective effect was smaller in both EO. The EOs prolonged the clotting time induced by L. muta muta venom and a procoagulant effect was observed on B. moojeni. In the comet assay, the flower EO presented anti-genotoxic action (damage frequency of only 11.6 - 34.9%) against the L. muta muta venom. The positive control (Doxorubicin) and the venom alone presented a damage frequency of 80.3% and 70.7%, respectively. The flower EO protected DNA from damage induced by L. muta muta venom. L. alba leaf and flower EOs presented anti-genotoxic action

Assays on DNA Damage and Repair in CLL.

Assays that measure DNA damage and repair are critical in evaluating the extent to which therapeutic agents damage DNA and in identifying whether DNA repair can limit the toxicity of chemotherapy. The COMET assays described in this guide should help readers evaluate single and double-strand breaks cause by chemotherapeutic agents and also monitor the ability of the cells to repair such damage. The EJDR assay described is a valuable tool to assess the ability of drugs and DNA repair proteins to modulate DNA repair capacity. Finally, the immunofluorescence assay described should allow accurate assessments of DNA damage and the kinetics of repair as measured by Ɣ-H2AX foci. This procedure can also be used to mechanistically investigate the recruitment of specific DNA damage and repair proteins in CLL cells.

In Vitro Approaches for Assessing the Genotoxicity of Nanomaterials.

Genotoxicity is associated with serious health effects and includes different types of DNA lesions, gene mutations, structural chromosome aberrations involving breakage and/or rearrangements of chromosomes (referred to as clastogenicity) and numerical chromosome aberrations (referred to as aneuploidy). Assessing the potential genotoxic properties of chemicals, including nanomaterials (NMs), is a key element in regulatory safety assessment. State-of-the-art genotoxicity testing includes a battery of assays covering gene mutations, structural and numerical chromosome aberrations. Typically various in vitro assays are performed in the first tier. It is not very likely that NMs may induce as yet unknown types of genotoxic damage beyond what is already known for chemicals. Thus, principles of genotoxicity testing as established for chemicals should be applicable to NMs as well. However, established test guidelines (i.e., OECD TG) may require adaptations for NM testing, as currently under discussion at the OECD. This chapter gives an overview of genotoxicity testing of NMs in vitro based on experiences from various research projects. We recommend a combination of a mammalian gene mutation assay (at either Tk or HPRT locus), the in vitro comet assay, and the cytokinesis-block micronucleus assay, which are discussed in detail here. In addition we also include the Cell Transformation Assay (CTA) as a promising novel test for predicting NM-induced cell transformation in vitro.

Single cell HaloChip assay on paper for point-of-care diagnosis.

This article describes a paper-based low cost single cell HaloChip assay that can be used to assess drug- and radiation-induced DNA damage at point-of-care. Printing ink on paper effectively blocks fluorescence of paper materials, provides high affinity to charged polyelectrolytes, and prevents penetration of water in paper. After exposure to drug or ionizing radiation, cells are patterned on paper to create discrete and ordered single cell arrays, embedded inside an agarose gel, lysed with alkaline solution to allow damaged DNA fragments to diffuse out of nucleus cores, and form diffusing halos in the gel matrix. After staining DNA with a fluorescent dye, characteristic halos formed around cells, and the level of DNA damage can be quantified by determining sizes of halos and nucleus with an image processing program based on MATLAB. With its low fabrication cost and easy operation, this HaloChip on paper platform will be attractive to rapidly and accurately determine DNA damage for point-of-care evaluation of drug efficacy and radiation condition. Graphical Abstract Single cell HaloChip on paper.

CometChip: a high-throughput 96-well platform for measuring DNA damage in microarrayed human cells.

DNA damaging agents can promote aging, disease and cancer and they are ubiquitous in the environment and produced within human cells as normal cellular metabolites. Ironically, at high doses DNA damaging agents are also used to treat cancer. The ability to quantify DNA damage responses is thus critical in the public health, pharmaceutical and clinical domains. Here, we describe a novel platform that exploits microfabrication techniques to pattern cells in a fixed microarray. The 'CometChip' is based upon the well-established single cell gel electrophoresis assay (a.k.a. the comet assay), which estimates the level of DNA damage by evaluating the extent of DNA migration through a matrix in an electrical field. The type of damage measured by this assay includes abasic sites, crosslinks, and strand breaks. Instead of being randomly dispersed in agarose in the traditional assay, cells are captured into an agarose microwell array by gravity. The platform also expands from the size of a standard microscope slide to a 96-well format, enabling parallel processing. Here we describe the protocols of using the chip to evaluate DNA damage caused by known genotoxic agents and the cellular repair response followed after exposure. Through the integration of biological and engineering principles, this method potentiates robust and sensitive measurements of DNA damage in human cells and provides the necessary throughput for genotoxicity testing, drug development, epidemiological studies and clinical assays.

Genotoxicity in gingival cells of patients undergoing tooth restoration with two different dental composite materials.

OBJECTIVES: Dental composite materials come into direct contact with oral tissue, especially gingival cells. This study was performed to evaluate possible DNA damage to gingival cells exposed to resin composite dental materials. MATERIALS AND METHODS: Class V restorations were placed in 30 adult patients using two different composite resins. The epithelial cells of the gingival area along the composite restoration were sampled prior to and after 7, 30, and 180 days following the restoration of the tooth. DNA damage was analysed by comet and micronucleus assays in gingival exfoliated epithelial cells. RESULTS: The results showed significantly higher comet assay parameters (tail length and % DNA in the tail) within periods of 30 and 180 days. The micronucleus test for the same exposure time demonstrated a higher number of cells with micronuclei, karyolysis, and nuclear buds. Results did not reveal any difference between the two composite materials for the same duration of exposure. CONCLUSION: Based on the results, we can conclude that the use of composite resins causes cellular damage. As dental composite resins remain in intimate contact with oral tissue over a long period of time, further research on their possible genotoxicity is advisable. CLINICAL RELEVANCE: Long-term exposure of gingival cells to two different composite materials demonstrated certain DNA damage. However, considering the significant decline in micronuclei frequency after 180 days and efficiency in the repair of primary DNA damage, the observed effects could not be indicated as biologically relevant.

Genotoxicidad sobre linfocitos humanos expuestos a PM10 de tres sitios del Valle de Aburrá (Antioquia) / Genotoxicity in human lymphocytes exposed to PM10 from three sites in the Valle de Aburrá (Antioquia)

Objetivo Evaluar la calidad del aire en tres sitios del Valle de Aburrá (Antioquia) a través de la determinación de la genotoxicidad del PM10 en linfocitos humanos. Métodos A partir del valor de referencia de PM10 para Colombia (50 μg/m3/año) se eligieron tres sitios del Valle de Aburrá con diferente promedio de PM10, Barbosa con 25 μg/m3, Corantioquia con 44 μg/m3y Facultad de Minas con 91 μg/m3. Los filtros de PM10 expuestos por 24 horas, se analizaron en la épocas de lluvia, transición y seca entre Julio de 2011 y abril de 2012. Con el extracto orgánico obtenido en cada filtro se trataron linfocitos humanos para evaluar in vitro el daño en el ADN por medio del ensayo Cometa Alcalino. Resultados Todos los sitios indujeron genotóxicidad altamente significativa (p< 0.001) respecto al control negativo, presentando el mayor daño durante la época de transición. Se observaron diferencias significativas (p<0.05) en la genotoxicidad inducida por el PM10 entre los tres sitios evaluados. Conclusiones Aunque los valores de PM10 reportados para Barbosa y Corantioquia fueron inferiores al nivel máximo permisible, se encontró actividad genotóxica del PM10 proveniente tanto de ellos como de Facultad de Minas cuyo valor de PM10 es superior a la norma. Estos resultados indican que únicamente el monitoreo fisicoquímico del material particulado es insuficiente para evaluar el riesgo relativo sobre la población expuesta. En consecuencia, estos estudios deben ser complementados con biomarcadores de genotoxicidad como el ensayo cometa.(AU)

Objective Assessing air quality by determining PM10 genotoxicity in human lymphocytes at three locations in the Valle de Aburrá (Antioquia department). Methods Three sites were chosen in the Valle de Aburrá (Barbosa, Corantioquia and the School of Mines) using Colombian reference (50 g/m3) and PM10content values, having annual low (25 mg /m3), medium (44 ug/m3) and high PM10 average (91 ug/m3). PM10filters were analyzed during three different seasons between 2011 and 2012: rainy, transitional and dry. Human lymphocytes were treated with the organic extract obtained from each filter to evaluate DNA damage using an alkaline comet assay. Results Genotoxicity was found to be highly significant (p<0.001) in all cases, compared to the negative control. The highest damage (six times) was seen in material from the School of Mines and during the transitional period. Differences were found between material from Barbosa and Corantioquia regarding that from the School of Mines concerning the induction of damage. Conclusions Although the PM10 values reported for Barbosa and Corantioquia were below the maximum permitted level, genotoxic activity was found for PM10from both sites as well as for the School of Mines. These results show that physical-chemical monitoring of particulate matter is not enough for assessing the exposed population's relative risk. Such analysis should thus be accompanied by using genotoxicity biomarkers, such as the comet assay.(AU)

Single cell trapping and DNA damage analysis using microwell arrays.

With a direct link to cancer, aging, and heritable diseases as well as a critical role in cancer treatment, the importance of DNA damage is well-established. The intense interest in DNA damage in applications ranging from epidemiology to drug development drives an urgent need for robust, high throughput, and inexpensive tools for objective, quantitative DNA damage analysis. We have developed a simple method for high throughput DNA damage measurements that provides information on multiple lesions and pathways. Our method utilizes single cells captured by gravity into a microwell array with DNA damage revealed morphologically by gel electrophoresis. Spatial encoding enables simultaneous assays of multiple experimental conditions performed in parallel with fully automated analysis. This method also enables novel functionalities, including multiplexed labeling for parallel single cell assays, as well as DNA damage measurement in cell aggregates. We have also developed 24- and 96-well versions, which are applicable to high throughput screening. Using this platform, we have quantified DNA repair capacities of individuals with different genetic backgrounds, and compared the efficacy of potential cancer chemotherapeutics as inhibitors of a critical DNA repair enzyme, human AP endonuclease. This platform enables high throughput assessment of multiple DNA repair pathways and subpathways in parallel, thus enabling new strategies for drug discovery, genotoxicity testing, and environmental health.

The determination of earthworm species sensitivity differences to cadmium genotoxicity using the comet assay.

The concept of species sensitivity differences is important in ecotoxicology and environmental risk assessment, but testing usually focuses on lethality of toxicants. The effects on the suborganismal level are mostly ignored; therefore, the present study assessed a biomarker of genotoxicity (the alkaline comet assay) to compare species sensitivities. Five earthworm species (Amynthas diffringens, Aporrectodea caliginosa, Dendrodrilus rubidus, Eisenia fetida and Microchaetus benhami) were exposed for 48 h to sublethal concentrations of cadmium sulphate in reconstituted soil water and DNA integrity was evaluated with the parameter Tail DNA %. Significant amounts of DNA damage were detected in three (A. caliginosa, D. rubidus and E. fetida) species. E. fetida exhibited the highest level of DNA damage, although D. rubidus showed the highest increase (3-fold) in DNA damage from the control. All exposed earthworms accumulated Cd, although body loads did not correspond with DNA damage levels; most of the Cd was probably sequestrated and rendered harmless.

Alpha-particle radiobiological experiments using thin CR-39 detectors.

The present paper studied the feasibility of applying comet assay to evaluate the DNA damage in individual HeLa cervix cancer cells after alpha-particle irradiation. We prepared thin CR-39 detectors (<20 microm) as cell-culture substrates, with UV irradiation to shorten the track formation time. After irradiation of the HeLa cells by alpha particles, the tracks on the underside of the CR-39 detector were developed by chemical etching in (while floating on) a 14 N KOH solution at 37 degrees C. Comet assay was then applied. Diffusion of DNA out of the cells could be generally observed from the images of stained DNA. The alpha-particle tracks corresponding to the comets developed on the underside of the CR-39 detectors could also be observed by just changing the focal plane of the confocal microscope.

Single-cell gel electrophoresis assay monitors precise kinetics of DNA fragmentation induced during programmed cell death.

BACKGROUND: Single-cell gel electrophoresis, or the comet assay, a technique widely used for DNA damage analysis, has been used recently for detecting DNA fragmentation in cells undergoing apoptosis. However, the number of variants of this assay used thus far primarily detected the late stages of DNA fragmentation. Therefore, monitoring the progression of DNA fragmentation, which could greatly improve the analysis of cell death induction and progression at the single-cell level, has not been possible with this assay. METHODS: In the present study, a modification of the original neutral comet assay developed by Ostling and Johanson (Biochem Biophys Res Commun 123:291-298, 1984) was used to detect various stages of DNA fragmentation. This assay involves cell lysis with anionic detergents at nearly neutral pH (9.5) and does not include high salt concentration, unlike most other published methods. BMG-1 human glioma cells were induced to undergo programmed cell death by treating with a large dose (100 microM) of etoposide, and comets were prepared after different durations (1-24 h) of treatment. RESULTS: In contrast to results of previously published studies, comets with different shapes reflecting progressive stages of DNA fragmentation were observed. Of these, six distinct shapes were identified and divided into three different categories based on the extent of fragmentation. Type A comets had a large head separated by a narrow "neck" region from an oval bulging tail that indicated initiation of fragmentation. Type B and C comets had a constantly diminishing head associated with a corresponding expansion of the tail and reflected intermediate and late stages of fragmentation, respectively. Type A and B comets appeared at a high frequency during early time points (1-6 h), whereas type C comets that indicated late stages of fragmentation were observed only after extended treatment (24 h). As a result, an elaborate kinetics of the progression of DNA fragmentation could be obtained. CONCLUSION: The present single-cell gel electrophoresis assay offers a significant improvement in monitoring the kinetics of DNA fragmentation induced during programmed cell death. Coupled with its simplicity and the ability to detect responses of small cell subpopulations, this method can be used for a reliable and sensitive analysis of the progression of cell death in different cell types and treatment conditions.

Microfabricated system for parallel single-cell capillary electrophoresis.

Performing single-cell electrophoresis separations using multiple parallel microchannels offers the possibility of both increasing throughput and eliminating cross-contamination between different separations. The instrumentation for such a system requires spatial and temporal control of both single-cell selection and lysis. To address these problems, a compact platform is presented for single-cell capillary electrophoresis in parallel microchannels that combines optical tweezers for cell selection and electromechanical lysis. Calcein-labeled acute myloid leukemia (AML) cells were selected from an on-chip reservoir and transported by optical tweezers to one of four parallel microfluidic channels. Each channel entrance was manufactured by F2-laser ablation to form a 20- to 10-microm tapered lysis reservoir, creating an injector geometry effective in confining the cellular contents during mechanical shearing of the cell at the 10-microm capillary entrance. The contents of individual cells were simultaneously injected into parallel channels resulting in electrophoretic separation as recorded by laser-induced fluorescence of the labeled cellular contents.

High throughput Comet assay using 96-well plates.

The single-cell gel electrophoresis or Comet assay is becoming established as an industrial genotoxicity screening test. The aim of this study was to increase the throughput of compounds tested and to minimize the amount of test compound needed for an assay. We modified practical aspects of our standard protocol and designed an experimental procedure suitable for use with 96-well plates. By using a suspension culture rather than attached cells, the modified protocol enabled parallel testing of four compounds on a single microplate (10 duplicate concentrations per compound). A significant reduction in work time was achieved by replacing the previously used Trypan blue dye exclusion (TBDE) test by an automated measurement of ATP levels as the concurrent viability test. The rapid and easy to perform ATP test was carried out towards the end of the 3 h treatment. In this way we were able to select for further analysis and slide preparation only those concentrations which induced the desired range of cytotoxicity. The suitability of the modified test conditions and reproducibility of test results was demonstrated by results obtained with standard mutagens and eight drug candidates tested at various concentrations. In each case the results obtained with the standard and the modified protocols were comparable. By introducing the changes to our standard protocol, combined with automated image analysis, we were able to more than double our previous throughput.

Automatic analysis of slides processed in the Comet assay.

In recent years the Comet assay (or single cell gel electrophoresis assay) has been established as a rapid and sensitive method for the detection of DNA damage. For early genotoxicity screening of new chemical entities in industrial toxicology, the Comet assay is more and more used for assessment of the DNA damaging potential of a test compound. In order to increase compound screening throughput, we have established an image analysis system for fully automated measurement of microscope slides processed in the Comet assay. For the comparative investigation various cell types, such as V79 Chinese hamster cells, mouse lymphoma cells and human leukocytes, were treated with several test compounds. Using tail moment as the quantitative parameter for comet formation, we show a very high correlation between our automatic image analysis system and a commercially available, interactive system (Comet Assay II of Perceptive Instruments). The possibility of analyzing 50 samples within 1 day and the high reproducibility of results make automated image processing a powerful tool for automatic analysis of slides processed in the Comet assay.

Cytometric methods to analyze ionizing-radiation effects.

Four cytometric assays for the assessment of radiation-induced DNA damage in individual cells are presented. Two of these, the alkaline and neutral comet assays, are useful for the detection of DNA damage due to very low radiation doses and promise to be useful for the quantitation of genomic damage after clinically or environmentally relevant exposures. The other two, the halo and halo-comet assays, reveal aspects of chromatin structure in the presence of DNA damage that reflect differences in intrinsic cellular radiosensitivity. Further development of these assays used alone, or in combination, should eventually lead to the definition of readily measurable cytometric parameters that will be useful as predictive markers for cellular responses to DNA damaging agents.

High resolution confocal laser scanning microscopy analysis of DNA excision repair capability in small volume marrow samples exposed to DNA directed treatment moieties.

BACKGROUND: Assessment of resistance to drug moieties in tissue culture is complicated by limited sample, clonal selection and alteration of cycling fraction and cycle duration in clonally mixed lesions. DNA damage assessment by single cell gel electrophoresis (SCGE) of excised DNA is limited by non-linear analysis in fluorescent light microscopy. Confocal Laser Scanning Microscopy (CLSM) with high N.A. magnification allows for quantitation of total excised DNA fragment size distribution but is still limited by the large volume required for labour intensive SCGE, precluding multi-exposure clinical testing. AIMS: To optimise sample requirement for SCGE and CLS. METHODS: Standard slide mounted bed gels were punched with multiple coded 6 mm wells and filled with suspensions of cells subjected to drug/concentration variations. After SCGE, 30 microns frozen sections were prepared of each well and mounted in ethidium bromide solution on multi-well hydrophilic slides to allow for short working distance of high resolution CLSM in a Zeiss Axiovert L410 SM. Testing for feasibility, reproducibility and consistency used both cultured standard leukaemic cell lines, normal human control marrow and clinical samples. RESULTS AND CONCLUSION: Multiple well SCGE followed by frozen section, high resolution CLSM allows for rapid analysis of high numbers of multiple drug exposure permutations clinically required.