Single Cell Gel Assay Research Articles

Alkaline single cell gel (comet) assay and genotoxicity monitoring using bullheads and carp

Monitoring genotoxicity of the environment using endemic organisms as sentinels requires the development of sensitive assays. Toward this end, we explored the feasibility of applying the alkaline single cell gel (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. Tail length has been correlated with level of genotoxicant exposure in a number of organisms. The fish used in this study were benthic feeding bullheads (Ameiurus nebulosus) and carp (Cyprinus carpio). On electrophoresis of erythrocyte DNA under alkaline conditions, we found a linear increase in the tail length/core width ratio over a broad range of cyclophosphamide doses. Freshly caught bullheads from seven different sites showed a wide range of DNA damage. Bullheads from Big Creek (western Lake Erie), Hamilton Harbour (western Lake Ontario), and the Detroit River gave ratios of 3.81 to 4.65. Based on polycyclic aromatic hydrocarbon (PAH) and polychlorinated biphenyl (PCB) levels, the sediment at these three sites is considered to be heavily polluted. Bullheads from southern Lake Huron, which is relatively clean, and from a fish hatchery in Brockport, New York, gave ratios between 1.30 and 1.40. Bullheads from Big Creek, maintained in the laboratory for 3 months, gave ratios which approached those seen in hatchery-bred fish. Results for carp were similar. Carp from Big Creek gave ratios of about 4.50, while carp from Lake Huron and laboratory-maintained carp gave values of 1.23 and 1.36, respectively. The results of the SCG procedure in bullheads and carp indicate that this assay is extremely sensitive and should be useful in detecting DNA damage caused by environmental contaminants.

Early effects of benzene exposure in mice. Hematological versus genotoxic effects.

Female BDF1 mice were exposed to 100, 300 and 900 ppm benzene 6 h/day, 5 days/week, up to 8 weeks. Hematological studies included peripheral blood data, T4 and T8 lymphocyte counts in the blood and the spleen, hemopoietic stem and progenitor cell assays in the marrow (CFU-S, CFU-C, BFU-E, CFU-E). The single cell gel assay ("comet assay") was applied in parallel with cells from the peripheral blood, bone marrow, spleen and liver. The results showed minor changes in the stem and progenitor cells and the development of a slight anemia at 4 and 8 weeks, in agreement with reported data. New was the increase of the T4/T8 ratio in the peripheral blood (not in the spleen) at the end of the first week of exposure to 300 and 900 ppm. The results of the "comet assay" indicate a much higher sensitivity to this test system (strand breaks and alkali labile sites of DNA). The tail moment indicative of the damage to DNA increased as early as 3 days with 300 ppm in the peripheral blood cells. Furthermore, the liver cells did react to a much higher extent than the other cells tested. With 100 ppm significant changes were seen in the liver after 5 days, but not in the blood. The repair, studied 24 and 48 h after the end of the exposure, was almost complete after 5-day exposure period in the blood and the liver, but not after 4 weeks of exposure with 300 ppm in the blood, and 100 and 300 ppm in the liver.

Video enhanced laser scanning microscopic analysis of DNA comet formation.

Laser Scanning Microscopy is a sensitive tool that provides a unique method of analyzing biological systems. Coupled with the Single Cell Gel assay, it allows for accurate and reproducible detection of DNA strand breaks. An understanding of the theory of DNA comet formation is lacking. Using dexamethasone induced apoptosis in murine thymocytes as a model for double strand breaks, we used video enhanced laser scanning microscopy to evaluate the leading edge of DNA migration in the single cell gel assay. In this system, comet length increases significantly within the first thirty seconds of electrophoresis, the greatest increase in length is completed within the first minute, and the first two minutes are important in significant increases in DNA migration during DNA comet formation.

Comparative investigations of the genotoxic effects of metals in the single cell gel (SCG) assay and the sister chromatid exchange (SCE) test

Sodium arsenite (NaAsO2) and cadmium sulphate (CdSO4) were tested for their ability to induce genotoxic effects in the single cell gel (SCG) assay and the sister chromatid exchange (SCE) test in human blood cultures in vitro. Both metals induced DNA damage in white blood cells that was expressed and detected as DNA migration in the SCG assay. Dose dependent effects were seen for cadmium in concentrations from 5 x 10(-4)-5 x 10(-3) M and for arsenic in concentrations from 2 x 10(-4)-1.5 x 10(-3) M. The distribution of DNA migration among cells, a function of dose, revealed that the majority of exposed cells expressed more DNA damage than cells from control cultures and that with increasing length of DNA migration the variability in migration among cells increased as well. Treatment of cells for 2 hr or 24 hr beginning 48 hr after the start of the blood cultures did not increase the SCE frequency in the case of cadmium but caused a small but significant SCE induction with arsenic at the highest concentration. The metal concentrations which could be investigated in the SCE test were much lower due to a strong toxic effect. Metal concentrations which were toxic in the SCE test were without visible effect in the SCG assay. Thus the two endpoints for the determination of genotoxic effects in vitro differed markedly with respect to the detection of genotoxicity induced by metals. These differences and the biological significance of the findings are discussed.

Evaluation of genotoxicity by DNA damages

A great number of genotoxins are known to be present in the environment. These genotoxins induce many kinds of DNA damage, and may cause changes in genetic information and cancer. Therefore, evaluation of such DNA damage is important to keep genetic information stable and to prevent cancer. We reviewed here methods used to assay the damage and the importance of this DNA damage in mutation. DNA damage is categorized into two groups, strand breaks and base modifications. To assay DNA strand breaks, the alkaline elution method, pulsed-field gel electrophoresis and single-cell gel assay are being used. The alkaline elution method determines both single- and double-strand breaks sensitively and quantitatively. Pulsed-field gel electrophoresis preferentially determines double-strand breaks, and the results of the method appeal to the eye as an electrophoretogram. The single-cell gel assay could determine both single- and double-strand breaks even in a single cell, and could evaluate susceptibility to the damage in individual cells. To assay base modifications, methods to detect differences in the physicochemical properties of the damage (physicochemical methods), immunoassays and the 32P-postlabeling method are being used. Physicochemical methods are suitable for chemically minor and abundant modifications such as those in 8-hydroxyguanine, using high-performance liquid chromatography or gas chromatography/mass spectroscopy. Immunoassays, by the use of specific antibodies against DNA damage, are highly sensitive to DNA damage such as changes in O6-methylguanine and thymine glycol, and simple once the assay systems have been established.(ABSTRACT TRUNCATED AT 250 WORDS)

Detection of apoptosis induced DNA cleavage in scrapie-infected sheep brain.

The pathogenesis and molecular basis of nerve cell death which accompanies scrapie infections in sheep are not well understood. Degeneration of neurons in culture caused by prion protein fragments has recently been reported to be consistent with mechanisms of cell death by apoptosis or programmed cell death. Apoptosis activation during prion-related encephalopathies has not yet been established in vivo. We report here the detection of DNA damage consistent with apoptosis in the brain cells of sheep infected with scrapie using laser scanning microscopic analysis of the single cell gel assay. We suggest that this DNA fragmentation is the result of the activation of the mechanisms characteristic of apoptotic cell death.

Determining DNA strand breaks using the laser scanning microscope

The single cell gel assay has been proven to be an effective, fast and accurate method of determining the amount of DNA single or double stranded breaks. The possible uses of this assay have already reached into some aspects of DNA damage caused by chemicals or other destructive agents. It has yet to reach even deeper into mutagenesis and carcinogenesis, both of which may show single or double stranded breaks to the DNA. Here we explore the effects that different concentrations of H2O2 have on the DNA of cells. The assay is performed by suspending cells in a low melt point agarose, spreading the agarose out on a frosted slide and letting it gel over ice. The cells are then treated with H2O2 to induce single strand DNA breakage. The cells are lysed in an alkaline environment, which separates the DNA into single strands and then electrophoresed. The gel is then stained with ethidium bromide.

Accurate Detection of DNA Single-stranded Breaks by Laser Scanning Microscopic Analysis of the Single Cell Gel Assay is Subject to Several Parameters

Accurate Detection of DNA Single-stranded Breaks by Laser Scanning Microscopic Analysis of the Single Cell Gel Assay is Subject to Several Parameters

DNA single strand breaks in human lens epithelial cells from patients with cataract.

In recent years, there has been renewed interest in the association between DNA damage to the lens epithelium and the development of lens opacities. Although a number of in vitro studies have indicated that lens epithelial cells are susceptible to a variety of DNA damaging insults and that these cells possess the capacity to repair such damage, no previous studies have directly addressed whether DNA damage is associated with human cataract in vivo. Utilizing samples of lens epithelial cells obtained from patients undergoing cataract surgery, the percentage of cells containing DNA single strand breaks was directly determined by the single-cell gel assay (SGA) method. Non-cataractous human Eye Bank lenses of similar ages to the cataractous samples and calf lenses were used as controls. In approximately 50% of the cataractous samples analyzed, the proportion of cells containing DNA single strand breaks was significantly higher than in control lenses. No relationship between age and DNA damage was noted. These findings are consistent with the hypothesis that in some human patients with cataract, DNA damage in the lens epithelial cell population may be related to the development of lens fiber cell opacity.

High-dose combination alkylating agents with autologous bone-marrow support in patients with breast cancer: preliminary assessment of DNA damage in individual peripheral blood lymphocytes using the single cell gel electrophoresis assay

The single cell gel (SCG) assay is a sensitive electrophoretic technique for detecting the presence of DNA single strand breaks and alkali-labile damage in individual cells. This technique was used to evaluate the levels of DNA damage in cryopreserved peripheral blood lymphocytes (PBLs) from 11 breast cancer patients treated with high doses of cyclophosphamide and cisplatin and provided autologous bone marrow transplantation after treatment. PBL specimens for the SCG study were obtained just prior to treatment, following the administration of cyclophosphamide and cisplatin for 2 days, and upon lymphocytic recovery. Based on a concurrent analysis of DNA damage in cryopreserved and noncryopreserved PBL samples from three patients, the mean level of DNA migration or the dispersion of damage among cells was not affected by the process of cryopreservation. The pre-treatment samples of several patients contained PBL with increased levels of DNA damage, presumably reflecting persistent DNA damage induced by previous treatment regimens. Chemotherapy resulted in a significant but variable increase in DNA damage in PBL samples from all patients. In this limited study, the level of damage did not correlate with serum levels of cyclophosphamide or with lymphocyte toxicity. Among the post-treatment samples, increased levels of DNA damage were absent in most but not all patients. The presence of damaged cells in the post-treatment samples may be indicative of an inadequate therapy regimen or of DNA damage resulting from non-therapy related processes. Because of its simplicity and short processing time, the SCG assay can be used to evaluate levels of DNA damage during the course of therapy, allowing the dose schedule to be altered to achieve a desired effect level.

Genotoxic activity of potassium permanganate in acidic solutions

Potassium permanganate (KMnO 4) combined with sulfuric acid is a strongly oxidizing mixture which has been recommended for the destruction and the decontamination of various mutagens/carcinogens in the publication series of the International Agency for Research on Cancer. Evaluation of the genotoxicity of 4 potassium permanganate solutions was performed using a microtechnique of the Ames test with the tester strains TA97, TA98, TA100 and TA102 with and without metabolic activation. Presence of direct-acting mutagens was detected in all the samples with the tester strain TA102 without S9 mix (163–357 revertants/μl of the solutions). Three samples containing either acetone or ethanol as an organic solvent also induced a mutagenic response on tester strain TA100 without S9 mix (167–337 revertants/μl). In addition, DNA damage in human peripheral blood lymphocytes was also measured for one of the mixtures by a new technique: the single-cell gel assay (SCGA). A sample with no organic solvent induced DNA damage in human lymphocytes with a dose-response relationship as determined by SCGA. The major mutagenic agent generated by the permanganate solutions was found to be manganese ion (Mn 2+). Both manganese sulfate (MnSO 4) and manganese chloride (MnCl 2) gave mutagenic dose-response relationships on tester strain TA102 without S9 mix. The mutagenic potencies were 2.8 and 2.4 revertant/nmole for MnSO 4 and MnCl 2 respectively. MnCl 2 also induced DNA damage in human lymphocytes as determined by the SCGA. The genotoxic effects of KMnO 4 in acidic conditions were probably mediated by the conversion of MnO 4 − to Mn 2+. KMnO 4 in alkaline solutions did not produce mutagenic species and may offer an alternative for the degradation of genotoxic compounds.