M.H.L. Green

The single cell gel electrophoresis assay (comet assay): A European review

The single cell gel electrophoresis (SCGE) assay is a rapid, simple, visual and sensitive technique for measuring DNA breakage in individual mammalian cells. Here we review the development of the SCGE assay (with particular reference to the alkaline version), existing protocols for the detection and analysis of comets, the relevant underlying principles determining the behaviour of DNA, and the potential applications of the technique.

Mutagen testing using TRP+ reversion in Escherichia coli

Escherichia coli strain WP2 and its repair-deficient derivatives are suitable strains for mutagen screening. In these strains, agents which cause base substitution mutations can be shown to increase the frequency of Trp+ revertants. In addition, agents causing many types of DNA damage can be detected through increased killing of the repair deficient derivatives. Four ways of performing tests are described: (a) Spot tests in which a small amount of the agent under test is placed directly on a selective agar plate. Trp+ revertants are counted and increased sensitivity of repair-deficient strains determined from the size of the zone of inhibition of cell growth. (b) Treat and plate tests, where a strain is treated with the agent under test and subsequently plated to determine survival or frequency of Trp+ revertants. (c) A simplified fluctuation test which shows exceptional sensitivity in measuring mutation with low levels of mutagens. (d) Use of a liver microsomal fraction in conjunction with treat and plate tests to detect metabolically activated mutagens. The merits and defects of these systems are discussed. Common pitfalls in evaluating tests and procedures for avoiding them are described.

Use of a simplified fluctuation test to detect low levels of mutagens

As a mutagen screening procedure we have used a modification of the Luria and Delbrück fluctuation test in which the individual tubes are scored by eye for the presence or absence of a mutation. The test is simple and extremely sensitive, detecting concentrations of mutagens up to 100-fold lower than conventional tests. Measuring mutation to tryptophan independence in Escherichia coli strain WP2 we have found that methyl methanesulphonate (0.5 mug/ml), mitomycin C (0.0015 mug/ml), dichlorvos (5 mug/ml, and K2CrO4 (0.5 mug/ml) are all positively mutagenic in the test, whereas NiCl2 is negative. Chronic exposure to low levels of mutagens using this method appears to induce more mutations than might be predicted by extrapolation from short exposure experiments at higher doses. The procedure is applicable to any system which involves mutation to prototrophy from a non-leaky auxotrophic requirement and should prove valuable in detecting and investigating the effects of low doses and chronic exposures.

UV-C sensitivity of unstimulated and stimulated human lymphocytes from normal and xeroderma pigmentosum donors in the comet assay : a potential diagnostic technique

We have studied incision-break formation in unstimulated and stimulated populations of human T-lymphocytes using the comet (single-cell microgel electrophoresis) assay. The frequency of strand breaks 1 h after UV-irradiation appears to be far greater in unstimulated than in stimulated lymphocytes from normal donors and the excess of strand breaks was observed for a far longer time after irradiation. This result corroborates the greater sensitivity of UV-C irradiation observed in a colony-forming assay but suggests that the defect may relate to a defect in strand rejoining rather than a defect in incision. Few strand breaks were seen in either unstimulated or stimulated lymphocytes of four xeroderma pigmentosum donors, suggesting that the method may offer a rapid diagnostic assay for XP.

Comparative Human Cellular Radiosensitivity: I. The Effect of SV40 Transformation and Immortalisation on the Gamma-irradiation Survival of Skin Derived Fibroblasts from Normal Individuals and from Ataxia-telangiectasia Patients and Heterozygotes

We have compared cell killing following 60Co gamma irradiation in 22 primary human fibroblast strains, nine SV40-immortalized human fibroblast lines and seven SV40-transformed pre-crisis human fibroblast cultures. We have examined material from normal individuals, from ataxia-telangiectasia (A-T) patients and from A-T heterozygotes. We have confirmed the greater sensitivity of A-T derived cells to gamma radiation. The distinction between A-T and normal cells is maintained in cells immortalized by SV40 virus but the immortal cells are more gamma radiation resistant than the corresponding primary fibroblasts. Cells transformed by plasmids (pSV3gpt and pSV3neo) expressing SV40 T-antigen, both pre- and post-crisis, show this increased resistance, indicating that it is expression of SV40 T-antigen, rather than immortalization per se which is responsible for the change. We use D0, obtained from a straight line fit, and D, estimated from a multitarget curve, as parameters to compare radiosensitivity. We suggest that both have their advantages; D0 is perhaps more reproducible, but D is more realistic when comparing shouldered and non-shouldered data.

Endogenous nitric oxide induced by interleukin-1β in rat islets of Langerhans and HIT-T15 cells causes significant DNA damage as measured by the ‘comet’ assay

We have used the comet assay (single cell gel electrophoresis) to measure nitric oxide‐induced DNA damage in rat islets of Langerhans and insulin‐containing HIT‐T15 cells. Damage was induced following treatment with the nitric oxide donor SIN‐1, which also releases Superoxide, but was not reduced by exogenous Superoxide dismutase, suggesting that nitric oxide itself, rather than Superoxide or peroxynitrite may be the active species. The DNA damaging effect of nitric oxide was easily detectable at the earliest time point tested (15 min). Damage also resulted following induction of nitric oxide synthase by the cytokine interleukin‐1β in both islets and HIT‐T15 cells and was prevented by replacing the substrate, arginine, with nitromonomethyl arginine. Thus intracellular levels of nitric oxide generated by interleukin‐1β‐induced nitric oxide synthase were sufficient to cause DNA damage in islet cells and HIT‐T15 cells.

A further assessment of factors influencing measurements of thioguanine-resistant mutant frequency in circulating T-lymphocytes

We have used the T-Lymphocyte cloning technique as a method of monitoring the human population for somatic cell mutant frequency. We present a statistical analysis of the experimental factors which may influence the observed mutant frequency. We have obtained consistently high plating efficiencies of T-cells from the mononuclear cell fraction from donor blood samples (mean of 56%, based on 123 observations from 70 individuals). Nevertheless, an inverse correlation of mutant frequency with plating efficiency was observed, and some experimental factors (serum and interleukin-2 batch, and worker) may have a significant effect on the observed mutant frequency. We discuss the difficulties that these possible effects present in establishment of a reference database and design of long-term studies. No significant effect of donor sex on mutant frequency was observed, but age (1.3% increase per year for normal adults) and smoking (56% increase over normal non-smokers) both significantly increased the mutant frequency. We discuss the utility of the assay for the monitoring of populations for heritable DNA damage, and we compare the results to those obtained with lymphocytes using other endpoints, e.g. chromosome aberrations, micronuclei and sister-chromatid exchange.

Repair-deficient bacterial strains suitable for mutagenicity screening: Tests with the fungicide captan

A spot test with selected repair-deficient strains of Escherichia coli is described which not only provides a sensitive assay for the mutagenic activity of chemicals, but also gives useful information about the characteristics of the mutagenic process. It has been shown that a substantial part of the mutagenic activity of the fungicide captan is due to excisable DNA damage mediated by a volatile breakdown product. The production of this volatile mutagen is greater at alkaline pH and is probably a result of hydrolysis. It may be very short-lived. Unlike other excisable DNA damage, that produced by the volatile mutagen does not depend on the ExrA+ and RecA+ repair functions for its mutagenicity. There is also a diffusible mutagen producing excisable damage the mutagenic action of which is Exr+-dependent in contrast to that of the volatile mutagen. It is suggested that a possible carcinogenic hazard might exist if captan is inhaled.

Tumor Necrosis Factor-α and Interferon-γ Inhibit Insulin Secretion and Cause DNA Damage in Unweaned-Rat Islets: Extent of Nitric Oxide Involvement

Nitric oxide has been implicated as one possible mediator of interleukin-1β (IL-1)-induced inhibition of insulin secretion and islet cell damage. The aim of this study was to define the effects of tumor necrosis factor-α (TNF) and interferon-γ (IFN) on nitric oxide production, insulin secretion, and DNA damage in islets from unweaned rats. Treatment of islets with 0.5–500 U/ml of either TNF or IFN on their own inhibited glucose-stimulated insulin secretion in a dose-dependent manner (minimum effective dose 5 U/ml). In combination, the cytokines exerted a pronounced synergistic inhibitory effect on secretion and were equipotent at causing a significant and concentration-dependent increase in culture medium nitrite levels, islet cyclic GMP formation, and DNA damage. Used alone or in combination, TNF and IFN significantly enhanced the activity of inducible nitric oxide synthase as determined by measuring the conversion of 14C-labeled arginine to 14C-labeled citrulline and nitric oxide. Use of arginine-free medium, without or with NG-monomethyl-L-arginine, resulted in inhibition of nitrite formation by 5–1,000 U/ml IFN + TNF and partial restoration of the insulin secretory response to glucose. Treatment of rat islets with increasing doses of TNF + IFN (5, 50, and 500 U/ml) resulted in a progressive increase in DNA damage, as shown by the comet assay, which detects DNA strand breaks in individual islet cells. The DNA damage caused by an intermediate concentration (50 U/ml) of TNF + IFN was comparable to that generated by IL-1 when used at 20 U/ml. We conclude that TNF and IFN induce nitric oxide formation, which partially inhibits glucose-induced insulin secretion and causes significant DNA strand breakage, but that as cytokine concentrations increase, non-nitric-oxide-mediated events predominate.

Comparative Human Cellular Radiosensitivity: II. The Survival Following Gamma-irradiation of Unstimulated (G0) T-lymphocytes, T-lymphocyte Lines, Lymphoblastoid Cell Lines and Fibroblasts from Normal Donors, from Ataxia-telangiectasia Patients and from Ataxia-telangiectasia Heterozygotes

We have measured clonal survival following gamma-irradiation of unstimulated (G0) T-lymphocytes from 35 donors, of 11 T-lymphocyte cell lines, of six lymphoblastoid cell lines, and of nine primary fibroblast strains for which we have G0 T-lymphocyte material from the same donor. Amongst the G0 lymphocytes we have results from nine normal donors, from eight cord bloods, from seven ataxia-telangiectasia (A-T) patients and from nine A-T heterozygotes. Although there is some variation between samples, G0 T-lymphocytes from normal donors appear to be slightly more radioresistant than T-lymphocyte lines, with a more shouldered survival curve. From our limited sample, lymphoblastoid cell lines appear to be slightly more radiosensitive than T-lymphocytes. The overall radiosensitivity of primary fibroblasts appears to be broadly similar to that of G0 T-lymphocytes. In nine instances, five A-Ts and four A-T heterozygotes, both G0 T-lymphocytes and primary fibroblasts from the same donor were tested. In five cases there was closely similar radiosensitivity in the two cell types, but in four cases there was some discrepancy. Further work, especially with normal donors, will be required in order to establish how reliably radiosensitivity in other cell types can be predicted from that of G0 T-lymphocytes. In all cell types the hypersensitivity of A-T cells was confirmed. Furthermore, the marginally greater sensitivity of A-T heterozygotes, when compared as a group with normals, was confirmed with G0 T-lymphocytes. Our results also suggest a slightly increased radiosensitivity in G0 T-lymphocytes from some, but not all, cord blood samples.