Effect of dose-rate and irradiation geometry on the biological response of normal cells and cancer cells under radiotherapeutic conditions.

Biological efficacy of radiation depends on its energy, dose, dose rate, and on the type of cell irradiated. Changes in the radiation-energy spectrum due to passage through absorbing and scattering media affect the variability of biological responses of the cells. We investigated the impact of photon-radiation dose rate on the biological response of both normal and cancer cells in culture exposed to radiation in various positions (relative to the axis of the radiation beam) and depth of the absorbing medium (water). Human cancer cells (A549 and HCT116) as well as normal human cells (BEAS-2B) were placed in a water phantom at different medium depths (3 cm, 15 cm) and exposed to 6-MV photon radiation delivered at a beam rate of either 100 or 600 MU/min (Monitor Units per minute). The applied dose was 5 Gy. Cells were exposed in the axis and four cm outside the radiation field. Radiation-induced genetic changes were estimated as frequency of micro-nucleated and apopototic-like cells, by use of a cytokinesis-block micronucleus test. A smaller dose rate induced more severe cytogenetic damage (formation of micro-nucleated and apoptotic cells) than a higher dose rate, both in normal and in cancer cells. More micro-nucleated and apoptotic cells were formed at larger depth than at smaller depth. This holds true for both the normal and the two types of cancer cell investigated. The extent of cytogenetic damage arising in cells placed outside the irradiation field is independent of positioning depth and dose rate. Exposure of cells to smaller dose rates and larger depths in water medium resulted in a better ratio of cytogenetic damage to cancer cells irradiated in the beam axis vs damage to normal cells exposed outside the radiation field.

Direct and bystander effects induced by scattered radiation generated during penetration of radiation inside a water-phantom.

In this study, the dose distribution of photon (6 MV) and electron (22 MeV) radiation in a water-phantom was compared with the frequency of apoptotic and micronucleated cells of two human cell lines (BEAS-2B normal bronchial epithelial cells and A549 lung cancer epithelial cells). Formation of micronuclei and apoptotic-like bodies was evaluated by the cytokinesis-block micronucleus test. Measurements were performed for five different phantom depths (3-20 cm). Irradiated cells were placed in a water-phantom in three variants: directly on the axis in the beam, under shielding (only in photon radiation) and outside the beam field. The results reveal a discrepancy between the distribution of physical dose at different depths of the water-phantom and biological effects. This discrepancy is of special significance in case of cells irradiated at a greater depth or placed outside the field and under shield during the exposure to radiation. The frequency of cytogenetic damage was higher than the expected value based on the physical dose received at different depths. Cells placed outside the beam axis were exposed to scattered radiation at very low doses, so we tested if bystander effects could have had a role in the observed discrepancy between physical radiation dose and biological response. We explored this question by use of a medium-transfer technique in which medium (ICM-irradiation conditioned medium) from irradiated cells was transferred to non-irradiated (bystander) cells. The results indicate that when cells were incubated in ICM transferred from cells irradiated at bigger depths or from cells exposed outside the radiation field, the number of apoptotic and micronucleated cells was similar to that after direct irradiation. This suggests that these damages are caused by factors released by irradiated cells into the medium rather than being induced directly in DNA by X-rays. Evaluation of biological effects of scattered radiation appears useful for clinical practice.

X-irradiation of human bronchial cancer cells causes the bystander effects in normal bronchial cells in vitro.

Using X radiation commonly used in radiotherapy of cancers we investigated bystander interactions between human cells: irradiated A549 bronchial carcinoma human cells and non irradiated BEAS-2B normal bronchial epithelial cells. Non irradiated cells were incubated in medium transferred from irradiated A549 cells (ICM-irradiation conditioned medium) for 48h and next the chromosomal damage and apoptosis were estimated. Conditioned medium collected from irradiated cancer cells induced in non irradiated cells of the same line as well as in BEAS-2B normal cells genetic changes such as micronuclei, chromatid and chromosomal breaks and condensation of chromatin characteristic for processes of apoptosis. Addition of only 1% of conditioned medium to fresh medium was sufficient to induction of bystander response to normal bronchial cells. The presented results in this study could have implications for human radiation risk and in evaluating the secondary effects of radiotherapy.

Effect of smoking and aging on micronucleus frequencies in human exfoliated buccal cells.

In the present work the frequency of micronuclei (MN) in exfoliated buccal cells in 120 healthy individuals with relation to sex, age and smoking was investigated. Neither age nor sex showed any effect on the level of micronuclei. Smoking has shown a significant effect upon basal DNA damage. In the present study the calculated background frequency of micronuclei (per mille) in oral epithelial cells of 50 smokers and 70 non-smokers were 1.50 (+/-0.47) and 0.55 (+/-0.32), respectively.

Antioxidant vitamins C, E and beta-carotene reduce DNA damage before as well as after gamma-ray irradiation of human lymphocytes in vitro.

The protective effect of Vitamins C, E and beta-carotene against gamma-ray-induced DNA damage in human lymphocytes in vitro was investigated. Cultured lymphocytes were exposed to increasing concentration of these vitamins either before or after irradiation with 2Gy of gamma-rays and DNA damage was estimated using micronucleus assay. A radioprotective effect was observed when antioxidant vitamins were added to cultured cells before as well after irradiation; the strongest effect was observed when they were added no later than 1h after irradiation. The radioprotective effect of vitamins also depended on their concentration; Vitamins C added at low concentration (1 microg/ml) before exposure of the cells to radiation prevented induction of micronuclei. Vitamin E at the concentration above 2 microg/ml decreased the level of radiation-induced micronuclei when compared to the cells irradiated without vitamin treatment. beta-Carotene was effective at all tested concentrations from 1 to 5 microg/ml and reduced the number of micronuclei in irradiated cells. The vitamins had no effect on radiation-induced cytotoxicity as measured by nuclear division index. The radioprotective action of antioxidant Vitamins C, E and beta-carotene was dependent upon their concentration as well as time and sequence of application.

The increment of micronucleus frequency in cervical carcinoma during irradiation in vivo and its prognostic value for tumour radiocurability.

A potential usefulness of micronucleus assay for prediction of tumour radiosensitivity has been tested in 64 patients with advanced stage (II B-IV B) cervical carcinoma treated by radiotherapy. The study of cellular radiosensitivity in vitro was conducted in parallel with the study of cellular damage after tumour irradiation in vivo. Radiosensitivity of in vitro cultured primary cells isolated from tumour biopsies taken before radiotherapy was evaluated using cytokinesis-block micronucleus assay. Frequency of micronuclei per binucleated cell (MN/BNC) at 2 Gy was used as a measure of radiosensitivity. Radiation sensitivity in vivo was expressed as per cent increment of micronucleus frequency in cells isolated from biopsy taken after 20 Gy (external irradiation, 10 x 2 Gy) over the pre-treatment spontaneous micronucleus level and was called MN20. Very low correlation (r = 0.324) was observed between micronucleus frequency in vitro and in vivo. Although micronucleus frequency at 2 Gy differed widely between tumours evaluated (mean MN/BNC was 0.224; range 0.08-0.416), no significant correlation was observed between this parameter and clinical outcome. The average increment of micronucleus frequency after 20 Gy amounted to 193% of spontaneous level (range 60-610%) and was independent of spontaneous micronucleation before radiotherapy. In contrast to in vitro results, these from in vivo assay seem to have a predictive value for radiotherapy of cervix cancer. The micronucleus increment in vivo that reached at least 117.5% of pretreatment value (first quartile for MN20 data set) correlated significantly with better tumour local control (P < 0.008) and overall survival (P < 0.045). Our results suggest that evaluation of increment of micronucleus frequency during radiotherapy (after fixed tested dose of 20 Gy) offers a potentially valuable approach to predicting individual radioresponsiveness and may be helpful for individualization of treatment strategy in advanced stage cervical cancer.

Modifying effect of vitamins C, E and beta-carotene against gamma-ray-induced DNA damage in mouse cells.

The modifying effect of treatment with vitamins C, E and beta-carotene on the clastogenic activity of gamma rays was investigated in mice. Damage in vivo was measured by the micronucleus assay in bone marrow polychromatic erythrocytes and exfoliated bladder cells. The vitamins were administered orally, either for five consecutive days before or immediately after irradiation with 2 Gy of gamma rays. The results show that pretreatment with vitamin E (100-200 mg/kg/day) and beta-carotene (3-12 mg/kg/day) were effective in protecting against micronucleus induction by gamma rays. Vitamin C depending on its concentration enhanced the radiation effect (400 mg/kg/day), or reduced the number of micronucleated polychromatic erythrocytes (50-100 mg/kg/day). Such effect was weekly observed in exfoliated bladder cells. The most effective protection in both tissues was noted when a mixture of these vitamins was used as a pretreatment. Administration of the all antioxidant vitamins to mice immediately after irradiation was also effective in reducing the radiation-induced micronucleus frequency. The data from the in vitro experiments based on the comet assay show that the presence of the vitamins in culture medium influences the kinetic of repair of radiation-induced DNA damage in mouse leukocytes.

[Vitamins as radioprotectors of normal cells]. / Witaminy jako radioprotektory komórek prawidlowych.

The radioprotective properties on antioxidants: vitamin C, vitamin E and beta-carotene are described in this work. Several problems concerning various biological actions of the antioxidants are presented.

Evaluation of frequency of micronuclei in exfoliated cells from bladder of mice treated with benzo(a) pyrene, 2-acetylaminofluorene and cyclophosphamide.

The micronucleus test (MNT) was applied in the epithelial cells from the bladder of mice treated with benzo(a)pyrene (BaP), 2-acetylaminofluorene (2-AAF) and cyclophosphamide (CP). Micronuclei (MN) were scored at different time on Feulgen and fast green stained smear preparations of exfoliated cells. These cells were obtained by scraping the internal surface of the bladder. The tested compounds gave the maximal response at 7-10 days after treatment. The number of MN in epithelial cells was dose dependent. These results suggest that exfoliated bladder cells from mice can be used as indicators for genotoxic damage in proliferating cell populations.

Mutagenic and clastogenic activity of the chloro-nitroimidazole radiosensitizer P40 in vitro and in vivo.

The hypoxic radiosensitizer 1-(2-hydroxy-3-methoxypropyl)-2-chloro-4-nitroimidazole [P40] was investigated for its mutagenic activity in bacterial Ames test as well as for genotoxic activity in micronucleus assay in vivo. This nitroimidazole showed the weak mutagenicity towards TA100 strain (base pair substitution) and towards TA98 strain (frameshift) only in the highest concentration. P40 induced also a significant increase in the frequency of micronucleated polychromatic erythrocytes (PCEs) at the doses of 0.6 mg/g and 1.2 mg/g. The maximum time response was at 48 h. The decrease of percentage of PCEs suggested the possible cytotoxicity on bone marrow cells after treatment with P40. Positive results in this battery short-term tests provide evidence of clastogenic activity of P40.

[Anemia and recurrences in acute lymphoblastic leukemia in children after the treatment and the frequency of the erythroblasts with micronuclei]. / Niedokrwistosci i wznowy szpikowe w ostrej bialaczce limfoblastycznej u dzieci po zakonczeniu leczenia a czestosc wystepowania erytroblastów z mikrojadrami.

A retrospective analysis of the relation of relapses and anaemias to the frequency of micronucleated erythroblasts in bone marrow smear in 140 children with acute lymphoblastic leukaemia after BFM and Memphis schemes of therapy was carried out. The anaemias correlated with the frequency of micronucleated erythroblasts in statistically significant manner. Correlation concerning relapses, was not shown. The frequency of micronucleated erythroblasts in children treated by BFM scheme is lower than by Memphis scheme. This suggests smaller genotoxicity of BFM scheme therapy.

Lack of genotoxic activity of metronidazole and P1 derivative in two eukaryotic tests.

Metronidazole [1-(2-hydroxyethyl)-2-methyl-5-nitroimidazole] and P1 derivative [1-(2-hydroxy-3-methoxypropyl)-2-methyl-4-nitroimidazole] were investigated for their genotoxic activity in two eukaryotic tests: mitotic recombination in yeast and micronucleus test in mice. Both compounds showed no genotoxicity in these eukaryotic assays contrary to their well-documented mutagenic activity in microbial short-term tests.