Comparison of alkaline single cell gel (Comet) and peripheral blood micronucleus assays in detecting DNA damage caused by direct and indirect acting mutagens.

The alkaline single cell gel (SCG) or 'comet' and peripheral blood micronucleus (pbMN) assay have been used to compare the effects of the direct acting mutagens, methyl methanesulfonate (MMS) and N-nitroso-N-methylurea (NMU), and the indirect acting mutagens, benzo[a]pyrene (BAP), cyclophosphamide (CP) 9, 10-dimethyl-1,2-benzanthracene (DMBA), and mitomycin C (MMC) in an inbred house mouse (Mus domesticus) strain. The alkaline SCG assay was able to detect DNA damage from direct acting mutagens. However, it appears that, even at the highest concentrations tested, the SCG assay was not able to detect DNA damage caused by 3 of 4 indirect acting mutagens tested. The exception was BAP. The pbMN assay was sensitive to DNA damage caused by both groups of mutagens. Multiple injections did not increase the sensitivity of the SCG assay to the indirect acting mutagen CP. Further, simultaneous injections of CP and MMS, in one experiment, resulted in significantly lower (p < 0.05) average DNA ratios and micronucleated polychromatic erythrocyte counts than those obtained after treatment with MMS alone. Although the SCG assay has been shown to be sufficiently sensitive to detect DNA damage caused by both direct and indirect acting mutagens in deermice (Peromyscus maniculatus) and bullheads (Ameiurus nebulosus), similar results are not seen in the inbred house mouse strain tested.

Comparison of DNA damage in peripheral blood and spleen lymphocytes using the single-cell gel assay.

The alkaline single-cell gel (SCG) or 'comet' assay has been applied to the detection of DNA damage from a number of chemical and biological factors in vivo and in vitro. In the past, a number of cell types has been used with peripheral blood lymphocytes being the most readily accessible. This study was designed to determine whether lymphocytes sequestered in the spleen might prove more sensitive to DNA damage than those in the peripheral circulation. This would result in a more effective SCG assay. Baseline DNA length to width ratios for peripheral blood and splenic lymphocytes did not differ significantly from each other (1.27 and 1.21, respectively). Neither did ratios of lymphocytes from the two sources, sampled 20 and 48 h after injection with 100 mg/kg methyl methanesulfonate (MMS) (3.81 and 3.62 at 20 h, respectively; and 1.96 and 2.21 at 48 h, respectively). Recovery from MMS damage at 168 h postinjection was also not different in the two groups of cells (1.13 and 1.16, respectively). However, an examination of cell profiles of DNA damage showed that splenic lymphocytes had a significantly higher percentage of damaged cells (63.33%) than did peripheral blood lymphocytes (40.67%) 48 h postinjection. Of the hypotheses proposed for this difference, the most likely seems to involve the different proportions of B- and T-lymphocytes present in the peripheral blood and the spleen. Since the difference between peripheral blood and splenic lymphocytes was seen only at 48 h postinjection, the use of splenic lymphocytes in the SCG assay is not advantageous under most circumstances.

Alkaline single-cell gel (comet) assay and genotoxicity monitoring using two species of tadpoles.

Small bodies of water (e.g., creeks, ponds, and drainage ditches) have received very little attention in genotoxicity studies, yet these areas are important because they are often the first to be affected by industrial effluents, sewage contaminants, accidental spills, internal combustion engine emissions, landfill runoffs, and pesticide uses. To address this deficiency, we examined erythrocytes in two species of tadpoles, Rana clamitans and Bufo americanus, using the alkaline single-cell gel (SCG) ("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. Exposure of R. clamitans todpoles to a range of concentrations of methyl methanesulfonate (MMS) produced a linear increase in DNA length to DNA core width ratios. This is consistent with findings in a number of other species. Time-dose experiments using MMS suggest that the peak level of DNA damage in R. clamitans todpoles occurred 42 hr after exposure. B. americanus tadpoles exposed to 6.25 mg/l of MMS for 12 hours had a significant increase in DNA damage over that seen in the controls. Freshly caught R. clamitans tadpoles from Highgate and B. americanus tadpoles from Duart, both on the north shore of Lake Erie, gave ratios of 2.78 and 2.07, respectively. This region of Ontario is a prime agricultural area and pesticide use is extensive. Tadpoles from Highgate and Duart, maintained in the laboratory for 4 months and 6 weeks, respectively, gave ratios of 1.29 and 1.44. The results of the SCG procedure in tadpoles indicate that this assay is extremely sensitive and suitable for detecting genotoxicity in the environment.

Comparison of three power supplies used for the single-cell gel assay.

The alkaline single-cell gel (SCG) assay, which determines DNA damage in mice treated with 100 mg/kg methyl methanesulfonate (MMS), was run by using three power supplies from Bio-Rad (models 3000Xi, 200/2.0, and Power Pac 300). Comparisons of the results obtained from the use of these power supplies showed differences in mean DNA tail length to width ratios and profiles of these ratios in individual cells. Model 200/2.0 power supply appeared to enhance significantly the sensitivity of the assay. In purchasing power supplies, there is a need to evaluate their effect on the sensitivity of the test.

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.