Nan Mei

Acute arsenite-induced 8-hydroxyguanine is associated with inhibition of repair activity in cultured human cells

8-Hydroxyguanine (8-OH-Gua) is one of the major modified bases in DNA produced by oxidative damage. Human lung carcinoma cells (A549) were treated with 0.5-2mM sodium arsenite for 4h. By an immunohistochemical type procedure, 8-OH-Gua was clearly detected in A549 cells using a fluorescence microscope and an increase in the percentage of A549 cells with oxidative DNA damage was observed using flow cytometry. The formation of 8-OH-Gua in DNA was also detected by a HPLC-ECD. A dose-dependent increase in oxidative DNA damage in A549 cells with increasing arsenite concentrations was obtained. Therefore, oxidative stress is induced after arsenite treatment. Furthermore, we also found that arsenite decreased the activity of the 8-OH-Gua repair enzyme, hOGG1 (8-oxoguanine-DNA glycosylase 1) as well as its gene and protein expression. We conclude that the 8-OH-Gua level in cultured human cells increases partly by the generation of reactive oxygen species (ROS) and partly by the influence on hOGG1 expression, followed by the inhibition of the repair activity for 8-OH-Gua.

Comparison of the frequency of T-cell receptor mutants and thioguanine resistance induced by X-rays and ethylnitrosourea in cultured human blood T-lymphocytes

We have investigated two assays for measuring the induction of mutations using human T-lymphocytes isolated from leukocyte residue buffy coats obtained from normal donors. Variant cell frequency of T-cells defective in the T-cell receptor (TCR) gene expression was measured using a 2-color flow cytometry, and 6-thioguanine-resistant (TGr) cells were determined using a cloning technique at the HPRT gene after treatment with 250 kVp X-rays or ethylnitrosourea (ENU). The frequencies of TCR mutant cells as well as those of TGr cells increased with increasing doses of X-rays or concentrations of ENU studied. For TCR mutants, the induced mutation frequencies at D37 (giving 37% survival) were 31.7 x 10(-4) and 11.0 x 10(-4) for X-rays and ENU, respectively. For TGr T-cells, the induced mutation frequencies at D37 for the same mutagens were 14.4 x 10(-6) and 75.5 x 10(-6), respectively. Over the dose range studied the relationship appears to be linear between the mutation induction of TCR and that of TGr for X-rays or ENU. However, X-rays may induce more TCR mutants against less induction TGr T-cells, and ENU may cause a reverse result. The sensitivity of the assay of each biological endpoint in human blood T-lymphocytes may be different.

Measurement of the CD3^-4^+ Variant T Cell Frequency by Flow Cytometry after X-Irradiation on Mice

Measurement of the CD3− 4+ Variant T Cell Frequency by Flow Cytometry after X‐ Irradiation on Mice: Naoki Kunugita, et al. Department of Radiation Biology and Health, School of Medicine, University of Occupational and Environmental Health, Japan—Variant cell frequency of mice T‐lymphocytes defective in the T‐cell receptor (TCR) gene expression was measured by flow cytometry. Splenic T‐lymphocytes were obtained from mice after irradiation and stained with fluorescein‐labeled anti‐L 3 T 4 (CD 4) and phycoerythrin‐ labeled anti‐CD3‐£ antibodies. They were analyzed with a flow cytometer to detect variant T cells lacking surface expression of TCR/CD 3 complexes which showed CD3− 4+ T cells. Variant T cells could be observed 3 days after 3 Gy irradiation. Frequency of variant T cells increased to the maximum level of 94×10−4 between 15 and 21 days after 3 Gy irradiation on BALB/c mice and gradually decreased with a half‐life of approximately 17 days. We examined the strain difference in radiosensitivity of splenic T‐lymphocytes in BALB/c, C57BL/6, C3H/ He and B6C3F1 mouse strains by this assay method. We observed that BALB/c showed the most radiosensitivity. This assay method is quick and easy in comparison with other methods and is considered useful for the mutagenicity test.

Influence of donor age on the cytotoxicity and mutagenicity of ethylnitrosourea in cultured human T-lymphocytes.

The effects of age on the cytotoxicity and mutagenicity of N-ethyl-N-nitrosourea (ENU) in human peripheral blood T-lymphocytes were investigated using colony-forming assay in vitro. ENU was shown to induce a dose-dependent increase in cell killing and in mutation frequencies (MF). No significant correlation between age and ENU-induced 6-thioguanine-resistant (TGr) MF at the hypoxanthine phosphoribosyl transferase (HPRT) locus of the X-chromosome was found after treatment with the same concentration of ENU (1 mM or 2 mM). There were also no significant differences among different donor age groups and the sensitivity parameters for exposure to ENU. As X-rays, the cytotoxic and mutagenic effects of ENU in cultured human T-lymphocytes appear not to be associated with age. These results suggest that the repair of mutagen-induced DNA lesions does not decline with age. Such knowledge has implications for risk assessment and protection against environmental mutagens.