Jillian E. Lowe

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.

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.

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.

A comparison of the agar cloning and microtitration techniques for assaying cell survival and mutation frequency in L5178Y mouse lymphoma cells

Microtitration methods for assaying cell survival and mutation frequency to ouabain resistance, 6-thioguanine resistance and 1-beta-D-arabinofuranosyl cytosine resistance in L5178Y mouse lymphoma cells were compared to the standard agar cloning technique. The two methods gave essentially similar results for untreated cells, and after treatment with ethyl methanesulphonate and 4-nitroquinoline 1-oxide. Potential advantages of the microtitration method as a routine assay system are discussed.

Correlation of UVC and UVB cytotoxicity with the induction of specific photoproducts in T-lymphocytes and fibroblasts from normal human donors.

Abstract— By using specific monoclonal antibodies in situ and a computer‐assisted image analysis system we have determined the relative induction of cyclobutane dimers, (6–4) photoproducts and Dewar isomers in human mononuclear cells and fibroblasts following irradiation with UVC, broad‐spectrum UVB and narrow‐spectrum UVB. The lamps produced these lesions in different proportions, with broad‐spectrum UVB inducing a greater combined yield of (6–4) photoproducts and Dewar isomers per cyclobutane dimer than UVC or narrow‐spectrum UVB. The relative induction ratios of (6–4) photoproducts compared to cyclobutane dimers were 0.15, 0.21 and 0.10 following irradiation with UVC, broad‐ or narrow‐spectrum UVB, respectively. Although Dewar isomers were induced by UVC, their relative rate of formation compared to cyclobutane dimers was significantly greater after irradiation with either broad‐spectrum or narrow‐spectrum UVB. These values were 0.001, 0.07 and 0.07, respectively. With each lamp source, we have determined the survival of normal human T‐lymphocytes and fibroblasts at fiuences, which induce equivalent yields of cyclobutane dimers, (6–4) photoproducts or (6–4) photoproducts plus Dewar isomers. Killing of fibroblasts appears to be associated with (6–4) photoproduct formation, whereas killing of T‐lymphocytes seems to be mediated by combined (6–4) plus Dewar yields. These results emphasize the need to study the biological effects of UVB because cellular responses may be different from those following UVC irradiation.

DNA strand breakage, cytotoxicity and mutagenicity of hydrogen peroxide treatment at 4°C and 37°C in L5178Y sublines

Abstract Cells from the L5178Y murine lymphoma subline LY-R are twofold more resistant to killing by ionizing radiation than the subline LY-S. In contrast, LY-R cells are more sensitive to killing by hydrogen peroxide. Cells of the two sublines in logarithmic growth phase were treated with hydrogen peroxide in phosphate-buffered saline for 1 h at 4°C or 37°C. From the comparison of D o values it followed that at 37°C LY-R were 3.6 times more sensitive to the killing effect of H 2 O 2 than LY-S cells; at 4°C they were 11 times more sensitive. Treatment with hydrogen peroxide at 4°C gave a considerable sparing effect, which was substantially greater for the LY-S subline; for LY-S cells D o was 5.7 times lower at 37°C than at 4°C, for LY-R cells only 1.9 times. The mutation frequency (HGPRT) in LY-R cells was increased in proportion to H 2 O 2 concentration and was the same at both treatment temperatures. In contrast, mutation frequencies initially increased, then decreased with increasing H 2 O 2 concentration in LY-S cells treated at 4 or 37°C. The concentration at which the decline was initiated was higher at 4 than at 37°C. DNA damage after H 2 O 2 treatment (both temperatures, 5 min) was estimated from the ‘comet’ assay (single-cell gel electrophoresis). The initial damage, but not the residual damage, differed significantly in LY sublines. A period of slower repair (between 3 and 10 min) was found in LY-R cells.

The mutagenic potency of 1,8-dinitropyrene in cultured mouse lymphoma cells

Although non-toxic, 1,8-dinitropyrene (1,8-DNP) was mutagenic for mouse lymphoma L5178Y cell when assayed for induced resistance to 6-thioguanine, methotrexate, ouabain and 1-beta-D-arabinofuranosyl cytosine. In bacteria, nitropyrenes are potent inducers of frame-shift mutations, and the induction of ouabain-resistant mutants, believed to be due to base-pair substitutions, suggests that the mechanism of action may be different in mouse cells and bacteria. Long treatment time were required to detect 1.8-DNP-induced mutants in L5178Y cells, suggesting the possibility of an inducible activation system. 4-Nitroquinoline 1-oxide was both toxic and mutagenic to these same 4 mutation assays after short (2h) treatment times. The dilemma that exists when comparing the mutagenic potential of test chemicals when concentration of mutagen, treatment times and toxicity are markedly different, is discussed.

COMET ASSAY TO DETECT NITRIC OXIDE-DEPENDENT DNA DAMAGE IN MAMMALIAN CELLS

Publisher Summary This chapter discusses the use of comet assay to detect nitric oxide-dependent DNA damage in mammalian cells. After hydrogen peroxide, nitric oxide (NO) is the most prevalent mutagen to which human DNA is exposed. NO may interact with cellular amines to form N-nitroso compounds. Its oxidation products nitrite and the higher nitrogen oxides also have biological activity. NO produce DNA strand breakage in mammalian cells by the DNA precipitation assay, in situ nick translation, and the comet assay. The comet assay is a sensitive method for the detection of DNA strand breaks in mammalian cell. The comet assay detects release of DNA from a highly supercoiled DNA-protein complex. In comparison with other sensitive methods, the comet assay is relatively robust and economical in its use of material. It has the specific advantage that as a single-cell assay, it can detect nonuniform response within a cell population, and characterize the behavior of different cell types within a mixed population. Only a small proportion of DNA-damaging agents, including ionizing radiation, bleomycin, and hydrogen peroxide, induces direct breakage of the DNA phosphodiester backbone.

Effect of deoxyribonucleosides on the hypersensitivity of human peripheral blood lymphocytes to UV-B and UV-C irradiation.

We have previously shown that non-cycling (unstimulated) human lymphocytes from normal donors show extreme hypersensitivity to UV-B irradiation, and are killed by an excisable lesion which is not a pyrimidine dimer or 6-4 photoproduct. In this paper we show that addition of the 4 deoxyribonucleosides to the medium, each at 10(-5) M, substantially increased the survival of non-cycling normal human T-lymphocytes following UV-B irradiation and substantially reduced the frequency of excision-related strand breaks in human mononuclear cells. Addition of ribonucleosides to the medium did not enhance excision-break rejoining. The survival of fibroblasts, of cycling T-lymphocytes and of unstimulated xeroderma pigmentosum T-lymphocytes was not enhanced by deoxyribonucleosides. This suggests that the hypersensitivity is due to reduced rejoining of excision breaks as a consequence of low intracellular deoxyribonucleotide pools and that it can be redressed by supplementation of the medium with deoxyribonucleosides or upregulation of ribonucleotide reductase following mitogen stimulation. We suggest that UV-B forms an additional DNA lesion which is not a pyrimidine dimer or 6-4 photoproduct, which is relatively common, and at which incision is particularly efficient. In fibroblasts, repair of this lesion is completed with high efficiency, whereas in normal unstimulated T-lymphocytes, rapid incision exacerbates the effects of the reduced rate of strand rejoining and leads to cell death.

Use of the comet assay to investigate possible interactions of nitric oxide and reactive oxygen species in the induction of DNA damage and inhibition of function in an insulin-secreting cell line.

We have previously used the comet assay to demonstrate that the nitric oxide donor 3-morpholinosydnonimine (SIN-1) produces DNA damage in rat islets of Langerhans and in the SV40-transformed insulin-secreting hamster cell line, HIT-T15. Damage is not prevented by the addition of superoxide dismutase (SOD). In the present study, we have compared SIN-1, which generates nitric oxide, superoxide anion and hydrogen peroxide, with two other nitric oxide donors, S-nitrosoglutathione (GSNO) and the tetra-iron-sulphur cluster nitrosyl, Roussins black salt (RBS). We have used the comet assay as a highly sensitive method to measure DNA-damaging ability, and also measured inhibition of DNA synthesis and inhibition of insulin secretion. We have examined the effect of SOD and catalase on each of these endpoints in HIT-T15 cells following a 30-min exposure to the compounds (24 h for DNA synthesis). All compounds produced a significant dose-dependent increase in strand-breakage formation and all inhibited DNA synthesis and glucose-stimulated insulin secretion. RBS was the most potent. SOD did not reduce the responses observed with any of the compounds. Catalase largely prevented DNA strand breakage, inhibition of DNA synthesis and inhibition of insulin secretion by SIN-1, but had no effect on responses to GSNO or RBS. Addition of SOD together with catalase gave no greater protection against SIN-1 than catalase alone. The nitric oxide and superoxide anion produced by SIN-1 are though to combine to form highly reactive peroxynitrite. In addition, H2O2 may be formed in the presence of SIN-1 and may form hydroxyl radical in the presence of a transition metal, such as Fe2+. It appears that in insulin-secreting cells, the effects of SIN-1 are largely mediated by this latter mechanism. In contrast, GSNO and RBS appear to act by a different mechanism, not overtly involving reactive oxygen species. GSNO and H2O2 show no significant interaction in the induction of DNA strand breaks. Both nitric oxide and H2O2 are effective, directly or indirectly, as DNA strand-breaking agents, inhibitors of DNA synthesis and inhibitors of insulin secretion.