The comet assay method: a new approach in genotoxicology research

The comet assay is a fast, simple and sensitive genotoxicological technique for measuring DNA damage in an individual cell of virtually any cell type of animal or plant origin. Electrophoresis of complete cell genome (assuming a comet-like shape) combined with the image analysis systems for comet analysis provide densitometric and geometric parameters describing the complete comet as well as the head and tail. The comet optical density values are used to quantify the total comet fluorescence and hence indicate DNA content and the level of damage. The application of this method in different fields makes it a powerful tool in human genotoxic study as well as in the estimation of environmental pollution.     

The "comet" assay for detection of potential genotoxicity of polluted water

The aim of this study was to determine the potential genotoxic activity of polluted water samples taken from wastewater from selected industrial plants in Kraków: 1. the Thermal-electric Power Station 2. the Institute of Metal Cutting. The recently developed single cell gel assay (SCG or comet assay), which is a quick and simple technique for the evaluation of DNA damage and repair in individual cells, was used. The assay was carried out on human hepatoma cells (Hep G2) as target cells. A greater number of cells with comets was observed in those treated in vitro with the polluted water samples (70%-88%) than in those in the control (22%, 33%). These preliminary results indicate that comet assay can have an application in biomonitoring studies for determining the potential genotoxicity of water pollutants.     

Development and validation of the in vivo alkaline comet assay for detecting genomic damage in marine flatfish

Biomonitoring is an important subject within environmental sciences. Biomonitoring tests are required to be quick, relatively inexpensive, accurate, and reproducible. No genetic test currently fulfils all of these requirements. The chromosome aberration and sister chromatid exchange tests are very time consuming, the DNA adduct technique is rather expensive, and the micronucleus test has not inconclusively proven its use as a reliable monitoring tool. This work is focused on the validation of the comet assay as a candidate for monitoring marine ecosystems. For the comet assay, this work deals with the effectiveness of tissue dissociation, storage of cells in lysing buffer and in liquid nitrogen, different electrophoretic conditions, neutralisation and fixation of slides, interindividual variation between samples, and responsiveness of four tissue types to ethyl methanesulphonate (EMS). The main conclusions are: (i) dissociation of solid tissues in a phosphate buffer supplemented with 200 mM N-t-butyl-alpha-phenylnitrone provides cells with an acceptable background DNA damage; (ii) freezing of cells or tissues in liquid nitrogen generally leads to an increase in DNA breakage, especially for liver, gill and kidney tissue; (iii) storage of slides in the lysing solution for up to one week gives minor changes in comet tails; (iv) differences in protocols for neutralisation and fixation may influence the results; (v) high intra- and interindividual variations in comets (length and DNA content) may obscure the interpretation of comet results; (vi) blood, gill, liver and kidney all showed a statistically significant increase of DNA damage after exposure to 50 mg EMS/l; (vii) electrophoresis at low voltage for longer periods is to be preferred to high voltage and short electrophoresis times. The simplicity and sensitivity of the comet assay make it an adequate test system for biomonitoring of chronic low level exposure. However, protocols and experimental conditions have to be chosen carefully.     

Disparity of serum transferrin receptor measurements among different assay methods

OBJECTIVE: To highlight differences in the quantification of transferrin receptor (TfR) concentration (a reliable index of iron deficiency) between three different assay methods. DESIGN: Methods comparison of TfR measurements in 'elevated' and 'normal' human sera using the Ramco, Quantikine and 'Lab' assays. SETTING: The Hospital for Sick Children, Toronto, Ontario, Canada. SUBJECTS: Pooled TfR for elevated and normal human sera obtained from the Ramco TfR assay kit. MAIN OUTCOME MEASURES: Differences between TfR concentrations in normal and elevated samples and repeatability for each assay method and limits of agreement in TfR quantification between assay methods. RESULTS: The mean TfR concentrations for the elevated reference serum samples was higher than the normal reference samples within each individual assay (P < 0.001); however, measurement agreement between methods was poor. CONCLUSIONS: Recognition of the relative differences in the values obtained from each of the assays should affect the interpretation of TfR concentration as an index of iron deficiency.     

Detection of DNA damage in stressed trout nucleated erythrocytes using the comet assay: protection by nitroxide radicals

Because previous literature reports have demonstrated that nucleated trout erythrocytes in conditions of oxidative stress are subjected to both membrane damage and a decrease in the enzymatic defense systems (glutathione peroxidase), which in turn lead to hemolysis, the present study was undertaken to determine whether DNA may be affected too, prior to the hemolytic event. Impairment of DNA in stressed trout erythrocytes was assessed using the comet assay--a rapid and sensitive, single-cell gel electrophoresis technique used to detect primary DNA damage in individual cells. In addition, indolinic and quinolinic nitroxide radicals were included in the study to determine their efficacy as antioxidants against free-radical-induced DNA damage. The parameters, tail length, tail intensity, and tail moment, used as an index of DNA damage, have shown that trout erythrocytes exposed to oxidative stress experience DNA damage prior to hemolysis and that the nitroxides significantly prevent this damage. This result provides further information about the potential use of these compounds as antioxidants in biological systems.     

DNA damaging effects of pesticides measured by the single cell gel electrophoresis assay (comet assay) and the chromosomal aberration test, in CHOK1 cells

One herbicide (isoproturon), two fungicides (carbendazim and chlorothalonil) and etoposide (an effective antitumor agent used as a positive control), were tested for their ability to induce cytotoxic and genotoxic effects in Chinese Hamster Ovary (CHOK1) cells. Etoposide induced DNA damage detectable both by the alkaline Single Cell Gel Electrophoresis (SCGE) assay and the chromosomal aberration (CA) test in absence of noticeable cytotoxicity. With the SCGE assay, a clear induction of DNA damage was observed for chlorothalonil within a 0.2 to 1 microM concentration range. In the CA test, chlorothalonil gave also positive results, inducing mainly chromosome breaks. In contrast, no DNA damage was observed with the SCGE assay for carbendazim and isoproturon. In the CA test, carbendazim induced only numerical aberrations in the concentration range of 25 microM to 100 microM, and isoproturon did not induce any significant increase in CA. In conclusion, chlorothalonil appears genotoxic in proliferative CHOK1 cells, and as expected, the aneugenic compound, carbendazim, did not induce DNA strand breaks in the SCGE assay.     

Benzene-induced genotoxicity in mice in vivo detected by the alkaline comet assay: reduction by CYP2E1 inhibition

Acute, severe injury of the rabbit spinal cord, induced by the weight-drop method, causes alterations of the enzyme activities related to cholinergic and energy metabolism. Morphological examinations at the trauma site show degenerative processes in neurons 0.5 hr posttrauma and a marked decrease in the number of living cells 24 hrs later. Both biochemical and cytochemical findings show that the tissue metabolic and morphologic derangement, caused by severe spinal cord injury, is mostly confined to the gray matter at an early stage (0.5 hr), whereas 24 hrs later the white matter is also involved. The decrease in choline acetyl-transferase and acetylcholinesterase activities in the gray matter parallels the impairment of complex IV (cytochrome c oxidase) of the respiratory chain and the presence of morphological alteration in neurons. The dramatic drop in the enzyme activities, observed 24 hrs after the induction of the severe trauma is clearly associated with the loss of cells.     

Caged amphibian tadpoles and in situ genotoxicity monitoring of aquatic environments with the alkaline single cell gel electrophoresis (comet) assay

In previous studies we demonstrated that indigenous amphibian tadpoles are suitable organisms for monitoring small bodies of water (e.g., creeks, ponds, and drainage ditches) using the alkaline single cell gel electrophoresis (SCG) or 'comet' assay. This approach involves detection, under alkaline conditions, of cell DNA fragments which on electrophoresis migrate from the nuclear core, resulting in a 'comet with tail' formation. However, although often plentiful, tadpoles are not present in all aquatic environments. Both larger bodies of water (e.g., lakes and rivers) and those impacted upon heavily by man (e.g., bodies of water near industrial sites, on landfills, and in urban areas) often do not support amphibian tadpole populations. An alternative approach to the collection of indigenous tadpoles is to place caged tadpoles at these sites for short term exposures to environmental contaminants. To determine the feasibility of such an approach, Rana clamitans (green frog) and Bufo americanus (American toad) tadpoles were housed in cages at 11 sites in southwestern Ontario (Canada). In a preliminary experiment, we found that tadpoles caged at a polluted reference site (Tallgrass Prairie Heritage Park in Windsor, Ontario) for either 7 or 14 days showed significant (P < 0.05) increases in DNA damage, relative to tadpoles caged in the laboratory in dechlorinated water. As a result we routinely used a 7 day exposure time. Significantly (P < 0.05) increased levels of DNA damage, relative to their controls, were observed in tadpoles caged at three sites along two creeks draining a large petrochemical installation south of Sarnia, Ontario; at two sites in the Tallgrass Prairie Heritage Park; and at a site along the Ecarte Channel which is part of the St. Clair River. The DNA damage levels of animals caged in Lake St. Clair, in the Trenton Channel of the Detroit River, at a landfill site, and in two creeks in the city of Windsor did not differ significantly (P > 0.05) from their controls. This study demonstrates that caged tadpoles are suitable for monitoring most bodies of fresh water, particularly those aquatic habitats mentioned above where indigenous tadpoles are not present. A combined approach of collecting indigenous tadpoles and using caged tadpoles should provide a sensitive system for aquatic genotoxicity monitoring.     

Developments in core analysis by NMR measurements

For a large suite of consolidated sandstone samples low in shale content we have measured the permeability k, irreducible water saturation Swi, porosity phi, electrical-resistivity formation factor F, porosity by NMR, geometric-mean relaxation times T1g, and stretched-exponential relaxation times T1s. We find that T1g (or T1s) is the decisive parameter for the estimation of k or Swi of porous sandstones by other than direct measurements of these quantities. The additional use of phi or F brings appreciable, but not decisive, improvement. We show isovalue maps of the error factor delta, which show substantial regions of near-minimum values of delta and show basic compatibility of our estimators for permeability with different published estimators. The exponents of T1g (or T1s) in our power-law estimators and those of various published estimators for k are not very far from 2.0 if either or both of phi and F are also used in the estimators.     

Chromosomal aberrations, sister-chromatid exchanges, cells with high frequency of SCE, micronuclei and comet assay parameters in 1, 3-butadiene-exposed workers

The association of occupational exposure to 1,3-butadiene (BD) and induction of cytogenetic damage in peripheral lymphocytes was studied in 19 male workers from a monomer production unit and 19 control subjects from a heat production unit. The exposure to BD was measured by passive personal monitors. The following biomarkers were used: chromosomal aberrations (CA), sister chromatid exchanges (SCE), cells with a high frequency of SCE (HFC), micronuclei, comet assay parameters like tail length (TL) and percentage of DNA in tail [T (%)] and polymorphisms of GSTM1 and GSTT1 genotypes. BD exposure with a median value of 0.53 mg/m3 (range: 0.024-23.0) significantly increased (a) the percentage of cells with chromosomal aberrations in exposed vs. control groups (3.11% vs. 2.03%, P<0.01), (b) the frequency of SCE per cell (6.96 vs. 4.87, P<0.001), and (c) the percentage of HFC (19.9% vs. 4.1%, P<0.001). BD exposure had no significant effects on formation of micronuclei and on comet assay parameters. Effect of smoking was observed only for HFC in BD-exposed group. GSTM1 genotype affected chromosomal aberrations in exposed group, while GSTT1 genotype affected chromosomal aberrations in controls. No effect of GSTM1 or GSTT1 genotypes was observed on any other biomarkers used.     

Evaluation of the in vitro direct and indirect genotoxic effects of cobalt compounds using the alkaline comet assay. Influence of interdonor and interexperimental variability

The mechanisms of cobalt-induced pulmonary interstitial fibrosis and cancer are incompletely understood. DNA damage, either induced by genotoxic (direct or via oxygen radicals) or co-genotoxic (e.g. inhibition of DNA repair) processes may play an important role in the initiation of cancer. The alkaline comet assay provides a sensitive tool to investigate these two processes. Cobalt metal, a mixture of cobalt with tungsten carbide and cobalt chloride, were compared for their DNA-damaging capacity. Concentrations from 0 to 6.0 microg Co-equivalent/ml were tested. All three compounds were able to induce DNA damage in isolated human lymphocytes from three donors, in a dose- and time-dependent way. A relatively large interexperimental and interdonor variability in response was observed. This was ascribed to technical parameters and unidentified individual factors. This confirms the importance of repeating experiments using the same and different donors. The DNA-damaging potential of the cobalt-tungsten carbide mixture was higher than that of cobalt metal and cobalt chloride, which had comparable responses. No significant increase of DNA migration was observed when the DNA of cells treated with cobalt metal, cobalt-tungsten carbide or tungsten carbide were incubated with the oxidative lesion-specific enzyme formamidopyrimidine DNA glycosylase. This suggests that during the short treatment period no substantial oxidative damage to DNA was produced. Cobalt metal was able to inhibit the repair of methylmethanesulphonate-induced DNA damage. This was concluded from simultaneous exposure to cobalt and methyl methanesulphonate, post-incubation and post-treatment with 1.2 microg/ml cobalt of methyl methanesulphonate-treated cells.     

Alloy phase analysis from measurements of bulk magnetic properties

Measurements of bulk magnetic properties were investigated to evaluate whether they can be used to reveal the microstructure and phase stability of alloys. Specifically, phase transformations in aluminum-copper alloys were followed with magnetic susceptibility measurements. The results suggest that bulk magnetic measurements can be used to predict microstructure and, thus, properties of alloys. The ability to characterize alloy properties and phase stability through correlation with electromagnetic measurements may allow significant improvements in the nondestructive evaluation of advanced alloy properties and the prediction of service life.     

Image analysis of naso-sinusal pathology with computerized tomography

Imaging analysis has been applied to study typical cases of nasosinusal diseases, in an attempt to determine its capabilities for CT scan interpretations. Thus, polyps with sinusitis, cerebrospinal fluid occupying sinuses, cysts and bone tumors may be compared in order to obtain objective results that may help in the differential diagnosis in these conditions.     

Image analysis to measure activity index of animals

The objective of the study was to present a method to quantify the behavioural response of animals to their micro-environment by using a camera system and a digitiser board. An algorithm was developed for analysing images and calculating activity, occupied zone and boundary of the animals. The developed method was tested on 3 different applications and animals. In the first application, the behavioural responses of broiler chickens to their thermal environment was measured. In the second application behavioural responses of pigs to their thermal environment were measured. In the third application, the response of water fleas to a chromium pollution was measured using the developed technique. Based on the experimental results, it can be concluded that the developed image analysis technique can be employed to quantify the behavioural responses of the tested animals to their micro-environment, in an easy and accurate way.     

Proteome analysis: biological assay or data archive?

In this review we examine the current state of proteome analysis. There are three main issues discussed: why it is necessary to study proteomes; how proteomes can be analyzed with current technology; and how proteome analysis can be used to enhance biological research. We conclude that proteome analysis is an essential tool in the understanding of regulated biological systems. Current technology, while still mostly limited to the more abundant proteins, enables the use of proteome analysis both to establish databases of proteins present, and to perform biological assays involving measurement of multiple variables. We believe that the utility of proteome analysis in future biological research will continue to be enhanced by further improvements in analytical technology.     

Dilatation measurements of plain carbon steels and their thermodynamic analysis

Dilatation of the low-carbon steels with small additions of mass contents of Mn (up to 1.50%), Si (up to 0.347%), Nb (up to 0.053%), and V (up to 0.082%), was measured at a heating rate of 3°C/min, and the experimental results were compared with calculations based on thermodynamic models. The differences between experiments and calculations were analysed. It was found that the thermal expansion of pearlite and of austenite in the steels exhibits almost linear temperature dependencies, and these dependencies are described very well by the present calculations. During the transformation of pearlite to austenite, contraction of the steels may occur due to the dissolution of cementite within a narrow temperature range. The dilatation of the steels during the transformation of ferrite to austenite depends on the competition between the thermal expansion and the transformation process, and it finally leads to an increase in the length change to a maximum followed by a decrease down to the temperature at which the transformation is completed. For some steels, however, a certain amount of ferrite may remain in the samples during heating even at temperatures well above that of the minimum dilatation. This will affect the determination of the A₃ temperature, and makes the expansion of the steels deviate from the true expansion behaviour of austenite.     

棒材加热炉热工测试与分析

对武钢棒材厂加热炉在普钢加热的条件下进行热工测试,结果表明,加热炉的热效率为45．41％,吨钢燃料消耗为1．455GJ。造成加热效率低、能耗高的主要原因有：重油雾化不充分、助燃空气预热温度低、排烟温度高、炉墙保温性能下降等。提出了在不进行大规模改造的条件下降低能耗和提高产量的主要措施,包括改善加热炉燃烧控制、促进助燃空气预热器的清灰、强化炉墙保温与炉内传热等。