Measurement of DNA strand breakage and DNA repair induced with hydrogen peroxide using single cell gel electrophoresis, alkaline DNA unwinding and alkaline elution of DNA

Three techniques: single cell gel electrophoresis (SCGE), alkaline elution of DNA (AE), and alkaline DNA unwinding (ADU) were chosen to compare the sensitivity among these methods in detection of DNA damage and repair in human diploid VH10 cell line after short-term exposure to hydrogen peroxide. Using SCGE technique a dose-dependent increase in DNA migration was found in cells exposed to hydrogen peroxide in concentration range from 10 micromol/l to 100 micromol/l. Alkaline DNA unwinding method detected increased level of single strand breaks (ssb) in concentration range from 25 micromol/l to 100 micromol/l of H2O2, and alkaline elution of DNA estimated increased DNA elution rate from concentration 50 micromol/l of H2O2. In a time course study to evaluate the kinetics of DNA repair, both SCGE and ADU techniques showed that the repair of DNA strand breaks is very rapid; the level of ssb in treated cells has returned to near the background level within two hours. After this time damage remaining in the DNA was in the form of oxidised bases as revealed the incubation of treated cells with specific DNA repair endonuclease, formamidopyrimidine-DNA glycosylase.     

Estimation of DNA single-strand breaks by single cells gel electrophoresis in tumor cells

Receptor-like protein tyrosine phosphatase beta (RPTP beta) shows structural and functional similarity to cell adhesion molecules (CAMs). It binds to several neuronal CAMs and extracellular matrix (ECM) proteins that combine to form cell-recognition complexes. Here, the authors discuss the implications of such complexes for intercellular signaling, and the regulation of RPTP activity by cell-cell and cell-ECM contact.     

A computer model for the analysis of DNA replication intermediates by two-dimensional agarose gel electrophoresis

We present a computer model to predict the patterns expected for the replication intermediates (RIs) of DNA fragments analyzed by neutral/neutral two-dimensional (2D) agarose gel electrophoresis. The model relies on the mode of replication (uni- or bi-directional), the electrophoretic mobility of linear DNA fragments and the retardation caused by the three-dimensional shape of non-linear molecules. The utility of this model is demonstrated with two examples: replication analysis of the plasmids pBR322 and pHH5.8 in Escherichia coli after digestions with EcoRI and HindIII, respectively.     

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.     

Apoptosis induced by X-rays and chemical agents in murine fibroblastic cell lines with a defect in repair of DNA double-strand breaks

This article describes the development and characteristics of a hospital-based lactation program and mother's own milk bank in a large pediatric hospital in the southwestern United States. Professional and technical staffing, physical space of the milk bank area, and the program's services and special features are outlined. Quality control issues about human milk preparation, fortification, storage, and transport are discussed.     

A standardized protocol for the rapid preparation of bacterial DNA for pulsed-field gel electrophoresis

A rapid method for the preparation of bacterial DNA for pulsed-field gel electrophoresis was developed for Gram-positive and Gram-negative bacteria. This method was accomplished by reducing the time for the cell lysis reaction, restriction endonuclease digestion, and electrophoresis to 1, 1.5, and 18 h, respectively. The whole procedure from the initial bacterial culture plate to the final analysis of restriction fragments can be completed within 24 h. This rapid method was successfully achieved for Staphylococcus aureus, Enterococcus faecalis, Neisseria gonorrhoeae, Salmonella typhimurium, Serratia marcescens, and Stenotrophomonas maltophilia.     

Use of alkaline comet assay (single cell gel electrophoresis technique) to detect DNA damages in lymphocytes of operating room personnel occupationally exposed to anaesthetic gases

Solid-phase microextraction (SPME), a new solvent-free sample preparation technique, was invented by C. Arthur and J. Pawliszyn in 1990. This method mainly was applied for the extraction of volatile and semi-volatile organic pollutants in water samples. However, since 1995, SPME has been developed to various biological samples, such as whole blood, plasma, urine, hair and breath, in order to extract drugs and poisons in forensic field. The main advantages of SPME are: high sensitivity, solventless, small sample volume, simplicity and rapidity. We have reviewed the papers published in recent years about SPME in biological samples, and sorted out main experimental conditions, such as fibers, matrixes, the extraction approaches and time, as well as the acceleration method. We would expect SPME technique to have a promising future for toxicological analysis in forensic practice.     

Colon-specific genotoxicity of heterocyclic amines detected by the modified alkaline single cell gel electrophoresis assay of multiple mouse organs

The in vivo genotoxicity of five heterocyclic amines-Trp-P-2 (13 mg/kg), IQ (13 mg/kg), MeIQ (13 mg/kg), MeIQx (13 mg/kg), and PhIP (40 mg/kg)-in the mucosa of gastrointestinal and urinary tract organs (stomach, duodenum, jejunum, ileum, colon, and bladder) was studied by the alkaline single cell gel electrophoresis (SCG) (Comet) assay. Male CD-1 mice were sacrificed 1, 3, and 8 h after intraperitoneal injection. All the heterocyclic amines studied yielded statistically significant DNA damage in the colon but not the small intestine (duodenum, jejunum, and ileum) or urinary bladder. In this study, five heterocyclic amines were injected intraperitoneally to avoid the consequences of ingestion. Thus, the extensive damage to colon DNA was concluded to be due, at least in part, to a systemic effect.     

Sensitivity of the denaturing gradient gel electrophoresis technique in detection of known mutations and novel Asian mutations in the CFTR gene

More than 500 mutations have been identified in the CFTR gene, making it an excellent system for testing mutation scanning techniques. To assess the sensitivity of denaturing gradient gel electrophoresis (DGGE), we collected a representative group of 202 CFTR mutations. All mutations analyzed were detected by scanning methods other than the DGGE approach evaluated in this study. DGGE analysis was performed on 24 of the 27 exons and their flanking splice site sequences. After optimization, 201 of the 202 control samples produced an altered migration pattern in the region in which an alteration occurred. The remaining sample was sequenced and found not to have the reported mutation. The ability of DGGE to identify novel mutations was evaluated in three Asian CF patients with four unknown CF alleles. Three novel Asian mutations were detected-K166E, L568X, and 3121-2 A-->G (in homozygosity)-accounting for all CF alleles. These results indicate that an optimized DGGE scanning strategy is highly sensitive and specific and can detect 100% of mutations.     

Characterization of the cell death process induced by staurosporine in human neuroblastoma cell lines

Staurosporine is a potent and non-specific inhibitor of protein kinases. There is also evidence of staurosporine being a potent inducer of apoptosis. In several human neuroblastoma cell lines (SH-SY5Y, NB69, IMR-5 and IMR-32) we have found 100 nM staurosporine to induce cell death in half the population (EC50). Electron microscopy of these cells, fluorescence microscopy after Hoechst-33258 staining of chromatin and agarose-electrophoresis of DNA, show two different types of cell death. SH-SY5Y and NB69 die by apoptosis and display all the characteristic features of it. IMR-5 and IMR-32 lack some of these features and a ladder pattern of DNA degradation is not found. Different morphological types of apoptosis have been described during the development of vertebrates; the possibility of finding a similar diversity in cell culture is suggested. On the other hand, staurosporine is a potent promoter of neurite outgrowth. In all the neuroblastoma cell lines we have tested, neurite-promoting and cell death-inducing staurosporine concentrations mostly overlap. This fact has not been reported before, probably because of an early versus late timing of these two different phenomena. The neuritogenic effect has prompted the suggestion that staurosporine could be a prototype of drugs for neurodegenerative diseases; the present study raises several concerns about such a proposal.     

A selective theory for the epigenetic specification of the monospecific antibody production in single cell lines

A new selective theory for the specification of the antibody production is presented. It is grounded on the following postulates: (a) the antigen stimulus triggers the synthesis of antibodies having a wide variety of specificities in a given immunocompetent cell and its progeny; (b) only these antibodies having affinity for the antigen have their synthesis stabilized, and competition between the diverse syntheses will favor the antibodies having the highest affinity; (c) there existes a threshold synthesis such that when the production of an antibody becomes lower than the threshold, it will go to zero. As a consequence, one should observe, immediately after the antigen stimulus, the transient appearance of a large number of seemingly unspecific immunoglobulins, followed by specific antibodies. The apparent binding constant of these antibodies should also increase during the specification period. Besides, one should observe a small, but detectable amount of self antibodies during the early period of specification. Finally, in the presence of several antigens, one should find cells which appear to produce antibodies against several antigens during a transient period.     

Ultraviolet radiation, but not gamma radiation or etoposide-induced DNA damage, results in the phosphorylation of the murine p53 protein at serine-389

Polyclonal antibodies were produced and purified that selectively react with a p53 epitope containing the murine phosphoserine-389 or the human phosphoserine-392 residue, but not the unphosphorylated epitope. These antibodies, termed alpha-392, were employed to demonstrate that the phosphorylation of this serine-389 residue in the p53 protein occurs in vivo in response to ultraviolet radiation of cells containing the p53 protein. After ultraviolet radiation of cells in culture, p53 levels increase and concomitantly serine-389 is phosphorylated in these cells. By contrast, the serine-389 phosphorylation of the p53 protein was not detected by these antibodies in the increased levels of p53 protein made in response to gamma radiation or the treatment of cells with etoposide. These results demonstrate an ultraviolet responsive and specific phosphorylation site at serine-389 of the mouse or serine-392 of the human p53 protein. Previous studies have demonstrated that this phosphorylation of p53 activates the protein for specific DNA binding. This study demonstrates in vivo a unique phosphorylation site in the p53 protein that responds to a specific type of DNA damage.     

DNA damage induced by m-phenylenediamine and its derivative in the presence of copper ion

We have previously reported that long-term priming of human polymorphonuclear neutrophilic granulocytes (PMN) with interferon-gamma (IFN-gamma) increased the fMLP-stimulated calcium influx. We now show that also after short-term incubation with IFN-gamma, PMN calcium metabolism is modulated. Single adherent cells in three different calcium-containing buffers (high, normal, and low [Ca2+]) were stimulated with the bacterial peptide fMLP or the Ca-ATPase inhibitor thapsigargin (Tg) after about 5 min preincubation with IFN-gamma. The results of this protocol indicated that IFN-gamma increases both calcium influx and calcium sequestration. Store dependent Ca2+ influx, directly measured on readdition of calcium to Tg-treated cells incubated in EGTA buffer, was significantly enhanced in IFN-gamma-treated cells. This effect of IFN-gamma was enhanced by the tyrosine kinase inhibitor herbimycin A. Strikingly, in low extracellular calcium concentrations, IFN-gamma induced calcium transients in 20%-60% of the cells. The proportion of PMN responding with Ca2+ transients increased with decreasing extracellular calcium concentration. Average lagtime from addition of IFN-gamma to a response that could be measured was 7.3 sec, and average increase in [Ca2+] above the basal level was 790 nM. These IFN-gamma-induced transients could not be depressed by herbimycin A. Thus, IFN-gamma can increase capacitative calcium influx, induce calcium transients, and possibly affect calcium sequestration in human PMN.     

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.     

Determination of cell fate by c-Abl activation in the response to DNA damage

The cellular response to DNA damage includes growth arrest and activation of DNA repair. Certain insights into how DNA damage is converted into intracellular signals that control the genotoxic stress response have been derived from the finding that the c-Abl protein tyrosine kinase is activated by ionizing radiation and other DNA-damaging agents. c-Abl associates with the DNA-dependent protein kinase (DNA-PK) and is activated by DNA-PK-dependent phosphorylation. The ataxia telangiectasia mutated (ATM) gene product also contributes to c-Abl activation. The demonstration that c-Abl binds to p53, induces the transactivation function of p53 and activates p21 expression has supported involvement of c-Abl in regulation of the p53-dependent G1 arrest response. Interaction between c-Abl and the Rad51 protein has also provided support for involvement of c-Abl in recombinational repair of DNA strand breaks. Defects in G1 arrest and repair predispose to replication of damaged templates and, in the event of irreparable DNA lesions, induction of apoptosis. The available evidence indicates that c-Abl effects a proapoptotic function by a mechanism largely independent of p53. c-Abl also functions as an upstream effector of the proapoptotic JNK/SAPK and p38 MAPK pathways. In addition, c-Abl-dependent inhibition of PI 3-kinase contributes to the induction of apoptosis. The findings thus suggest that, in response to genotoxic stress, c-Abl functions in determining cell fate, that is growth arrest and repair or induction of apoptosis. The physiologic function of c-Abl may reside in control of the cellular response to DNA strand breaks that occur during DNA replication, genetic recombination and gene rearrangements.     

Detection by denaturing gradient gel electrophoresis of an Arg1689Cys mutation in a Chinese patient with mild hemophilia A

OBJECTIVE: To examine the ease of endotracheal intubation on the ground for various rescuer positions. METHODS: Six female and 18 male emergency medical technicians were asked to intubate a Laerdal Megacode Trainer placed on the ground. Rescuers assumed the following positions in random order: prone, sitting, kneeling at the mannequin's head, and straddling the chest. The authors measured times 1) for changing from mask ventilation to assuming intubation position and 2) from touching the laryngoscope to putting it down. Incidences of esophageal tube placement and clicks (possible tooth damage) were noted. The rescuers rated their satisfaction with each position on a six-point scale (1 = very good, 6 = insufficient). Total intubation times of the other three positions were compared with that for prone by rank order test for paired observations. Handling, esophageal positions, and clicks of the other three positions were compared with those for prone by sign test for paired observations. A Bonferroni correction (factor 12) was applied. RESULTS: Mean total intubation times (in seconds) were 11.8 +/- 3.3 for prone, 13.9 +/- 4.7 for sitting, 11.4 +/- 4.5 for kneeling, and 16.2 +/- 5.8 for straddling. The difference between straddling and prone was statistically significant (p < 0.005). For handling, the results were for prone 3.0 +/- 1.4, for sitting 3.1 +/- 1.1, for kneeling 2.2 +/- 0.6, and for straddling 2.8 +/- 1.4. Esophageal positions occurred for prone 1, for sitting 1, for kneeling 2, and for straddling 3. Clicks were counted for prone 2, for sitting 1, for kneeling 1, and for straddling 0. CONCLUSIONS: All tested positions provide satisfactory conditions for intubation on the ground. The straddling position requires statistically, but not clinically, significantly more time for intubation than does prone and may be an important backup position if access from behind the patient's head is impossible.     

Protective effects of green tea on hepatotoxicity, oxidative DNA damage and cell proliferation in the rat liver induced by repeated oral administration of 2-nitropropane

To evaluate the benefit of green tea in mitigating hazards caused by repeated exposure of 2-nitropropane (2NP), we examined the effects of the tea on toxic indices, oxidative DNA damage and cell proliferation in the liver of 2NP-treated rats. Male Fischer 344 rats were administered, by gastric intubation, a total of six doses of 60 mg/kg 2NP(L), or alternatively two doses of 90 mg/kg and then four doses of 120 mg/kg 2NP(H) during 2 weeks. Green tea infusion was given to the rats as drinking water 1 week before the 2NP treatments and throughout the experiment. Significant elevation of hepatotoxic indices was evident in the 2NP(H)-treated group, such as an increase of serum glutamic-oxaloacetic transaminase (GOT) activity and of hepatic lipid peroxidation, together with a decrease in hepatic glycogen and serum triglyceride, and degenerative changes in the hepatocytes. A dose-related increase was observed in oxidative DNA damage and cell proliferation in the liver. Green tea effectively inhibited all of above changes induced by 2NP treatment, suggesting that tea intake may be effective for preventing the hepatic injuries after chronic exposure to 2NP.