Alba M. Güerci

Aneugenic effects of some metal compounds assessed by chromosome counting in MRC-5 human cells.

Development of a comprehensive test battery is necesary for the evaluation and detection of aneugenic chemicals. The chromosome couting method was used in the present study. The aneugenic ability of cadmium choride (1.0, 2.0 and 4.0x10(-3) mM), cadmium sulfate (3. 3, 6.7x10(-5) and 1.3x10(-4) mM), potassium dichromate (2.5, 5. 0x10(-4) and 1.0x10(-3) mM) and cacodilic acid (1.25, 2.5 and 5. 0x10(-2) mM) were analysed using MRC-5 cells which have a modal diploid number of 2 n=46 with a spontaneous aneuploid or polyploid cells not higher than 13% and 8%, respectively.All compounds induced significant increments of aneuploid cells in relation to negative controls. The frequency of aneuploid cells increased in all treatments with cadmium chloride. Cadmium sulfate induced significant increments of aneuploid cells with the two higher doses. All the doses of potassium dichromate increased the frequency of aneuploid cells although to a lesser degree than the other compounds. In these cases, differences were in the borderline of statistical significance (p<0.05). Moreover, a low number of cells could be analysed in treatments with the highest dose due to the decrease in the mitotic index. Results obtained are coincident with previous reports using the same methodology in the sense that induced aneuploidy was mainly evidenced by the increase of hypodiploid but not hyperdiploid cells. In addition, anaphase-telophase analysis of the effects of the same doses of these metal compounds in CHO cells showed significant increments of lagging chromosomes and increased frequencies of kinetochore positive micronuclei in MRC-5 cells. These findings could be considered as an indication that the main cause of unequal chromosome separation is the failure of kinetochores to attach the spindle apparatus either by alteration of its protein components or by the altered chromatid separation in anaphase.

Genetic instability induced by low doses of x-rays in hamster cells.

Purpose: Genomic instability involves time delayed events and can be manifested as elevated rates of heritable changes in the progeny of irradiated cells. To study the induction of chromosomal instability by very low doses of radiation Chinese Hamster Ovary (CHO) cells were exposed to 10 – 50 milisieverts (mSv) (≈10 – 50 miligrays (mGy)) of x-rays. Materials and methods: Control and irradiated cell populations were assayed for chromosomal aberrations and assessed using a micronucleus test and anaphase-telophase analysis at the first cell division post-irradiation and at every four population doublings thereafter up to 16 population doublings post-irradiation. Results: Frequencies of micronuclei, anaphase-telophase alterations and chromosomal aberrations were increased when the cells were analysed immediately after x-ray exposure. Micronuclei and anaphase-telophase alterations showed significantly increased frequencies when they were analysed at 12 and 16 population doublings after exposure to 50 mSv. Chromosomal aberrations increased significantly at 12 and 16 population doublings after exposure to 10 mSv and 50 mSv. Conclusions: Our results are consistent with the presence of a phenomenon by which the initial DNA damage in the surviving cells is memorized. Micronuclei and achromatic lessions were the main cytogenetic damage observed in cells exposed to very low doses of x-rays, indicating that these low doses are able to induce genetic instability.

DNA damage in Chinese hamster cells repeatedly exposed to low doses of X-rays

In a recent paper we reported the results of an experiment carried out by analysing chromosomal damage in Chinese hamster (CHO) cells exposed to low doses of X-rays. The present investigation was undertaken in order to validate those results using a different approach, the single cell gel electrophoresis assay (comet assay) immediately after irradiation. Cells were cultured during 14 cycles, irradiation treatment was performed once per cycle when the cells were at 90–95% of confluence. Doses of 2.5, 5.0 and 10.0 mSv were used. Sequential irradiation of CHO cells induced a decrease of cells without migration and an increase of cells showing DNA damage with the three doses employed. Significant increases of low-level damaged cells (p < 0.001) were found for the 14 exposures when compared to controls except for the first irradiations with 2.5 and 10 mSv, respectively. No significant increase of the frequency of cells with severe damage was observed in any case. These findings could be explained by assuming a complex interactive process of cell recovery, DNA damage and repair together with the induction of genomic instability, the incidence of bystander effects as well as some kind of radioadaptative response of the cells. If these phenomena are limited to the cell line employed deserves further investigation.

Differential response of two cell lines sequentially irradiated with low X-ray doses

An experiment was designed to compare the effect of repeated low doses of X-rays in two different cell lines: one transformed, epithelial like and aneuploid Chinese hamster ovary K-1 (CHO-K1); the other originated from a human primary culture, fibroblast, diploid and non-transformed, MRC-5. CHO and MRC-5 cells were cultured for 14 or eight passages, respectively. Irradiation was performed once per passage when cells were in the quiescent state (90 – 95% in G1/G0). Cells were exposed to 10.0 mSv X-ray doses. Ionizing radiation did not induce apoptosis or necrosis in the exposed CHO cell population. Significant increases of low-level damaged cells (degrees 1 and 2) were found for the 14 cycles of radiation when compared with controls, except for the first irradiation cycle. No significant increases in the frequency of cells with severe damage were observed. The frequency of MRC-5 cells with low-level damage increased significantly when compared with controls for radiation cycles seven and eight. Significant increases of apoptosis, necrosis and severe damage were found only for the highest dose. Transformed and non-transformed cell types responded differently to direct and indirect damage using low-dose repeat exposures to ionizing radiation. Though more investigation is needed to understand the mechanisms of radiation effects in chronic low-dose-exposed cell populations, cellular type should be taken into account in the design of in vitro experiments for understanding low-dose-irradiation effects.

Cytogenetic analysis in Chinese hamster cells chronically exposed to low doses of X‐rays

Purpose: It is important to develop simple experimental models to assess the induction of DNA damage and study the different factors involved under controlled conditions. This paper describes the cytogenetic analysis carried out in Chinese hamster cells (CHO) sequentially exposed to very low doses of X‐rays. Materials and methods: CHO cells were cultured for 14 passages. Irradiation treatment was performed once per passage, and three irradiation doses were employed: 2.5, 5.0 and 10.0 mSv. Results: Sequential irradiation of CHO cells did not increase the yield of chomatid‐ or chromosome‐type aberrations. However, a significant increase of achromatic lesions (gaps) was found after the first or second X‐ray dose, with all three irradiation doses employed. Conclusions: The variation in the frequency of gaps as well as that in the mitotic index during the 14 cycles of radiation could be an indication of the induction of genomic instability. According to this, continuous rises and falls in the frequency of gaps as well as in the mitotic index reflects the simultaneous induction of endogenous DNA damage, cell death and cell survival.

Assessment of genotoxic damage in lymphocytes of hospital workers exposed to ionizing radiation in Argentina.

The authors aimed to assess genotoxic damage in the lymphocytes of workers chronically exposed to ionizing radiation. The studied population included 15 exposed donors of the radiology unit of a public hospital in La Plata, Argentina. The control group included 15 nonexposed employers from administrative areas that the authors matched by age, sex, and smoking habits. The mean frequency of cytogenetic damage was higher in the exposed group than in the unexposed group for aneuploidy and structural chromosome aberrations. They observed the highest difference when achromatic lesions (or gaps) were considered. The comet assay showed that the frequency of cells with low damage was higher in the exposed group than in the unexposed group. A mean length analysis showed significant differences between exposed and nonexposed people. The results can be considered to be consistent evidence of occupational radiation exposure, and the results indicate that the workers must be advised to avoid or minimize their exposure.

Polymorphic Variants in Oxidative Stress Genes and Acute Toxicity in Breast Cancer Patients Receiving Radiotherapy.

Purpose Reactive oxygen species (ROS) are generated as an indirect product of radiation therapy (RT). Genetic variation in genes related to ROS metabolism may influence the level of RT-induced adverse effects. We evaluated the potential association of single nucleotide polymorphism (SNP)–related response to radiotherapy injury in breast cancer patients undergoing RT. Materials and Methods Eighty patients receiving conventional RT were included. Acute effects were evaluated according to the Radiation Therapy Oncology Group (RTOG) scores. DNA was extracted from blood and buccal swab samples. SNPs were genotyped for GSTP1, GSTA1, SOD2, and NOS3 genes by polymerase chain reaction–based restriction fragment length polymorphism. Univariate analysis (odds ratios [ORs] and 95% confidence interval [CI]) and principal component analysis were used for correlation of SNPs and factors related to risk of developing ≥ grade 2 acute effects. Results Sixty-five patients (81.2%) showed side effects, 32 (40%) presented moderate to severe acute skin toxicity, and 33 (41.2%) manifested minimal acute skin reactions by the end of treatment. In both univariate and multivariate analyses, nominally significant associations were found among body mass index (OR, 3.14; 95% CI, 8.5338 to 1.1274; p=0.022), breast size (OR, 5.11; 95% CI, 17.04 to 1.54; p=0.004), and grade ≥ 2 acute radiation skin toxicity. A significant association was also observed between NOS3 G894T polymorphism (OR, 9.8; 95% CI, 211.6 to 0.45; p=0.041) and grade ≥ 2 acute radiation skin toxicity in patients with neo-adjuvant chemotherapy treatment. Conclusion The analysis of the factors involved in individual radiosensitivity contributed to the understanding of the mechanisms underlying this trait.

Evaluation of radioadaptive response induced in CHO-K1 cells in a non-traditional model.

Purpose: The present study was designed to evaluate the effects of sequential exposures to low doses of gamma-radiation that induce a radioadaptive response to a later high-dose of radiation in CHO-K1 cells. Materials and methods: Cells were cultured in four dilution cycles and grown to confluency. Radiation treatment was performed once per cycle with 0.1 Gy gamma-rays. After the last radiation period (chronic radiation) the culture was irradiated with a higher dose (1 Gy). Each cell culture was immediately divided into two fractions: one of them was used to carry out the comet assay and the other for the structural chromosome aberration test. In the first fraction, genotoxic damage was evaluated by degree of damage in 300 cells per experimental point. The second assay was performed with 400 cells per treatment. The statistical analysis was carried out using the χ2-test. Results: Results from these assays demonstrated a genotoxic effect for both the adaptive and acute treatments (p < 0.001). The comet assay showed a significant increase in damage for the combined treatment when compared with 1 Gy treatment (p < 0.001). The frequency of chromosomal aberrations (CA) was lower for the combined treatment than for that using the highest radiation dose. Conclusions: These results suggest the possible induction of a radioadaptive response after the sequential exposure to very low doses of radiation. The finding of decreased cytogenetic damage after one cell cycle and not immediately after radiation could indicate the eventual potentiation of repair mechanisms.

Evaluation of resveratrol radiomodifying potential for radiotherapy treatment

Radiotherapy is widely used for cancer treatment. However, its adverse effects that may develop during the course of treatment have forced to search agents to protect biological systems against the deleterious effects of radiation. Resveratrol (3,4,5-trihydroxy-trans-stilbene; RSV) is a natural polyphenol currently promoted for its beneficial pleiotropic effects on health, which has been shown to exhibit antioxidant properties while inhibiting the growth of tumor cells. In radioresistant tumors, RSV could contribute to reduce recurrence and treatment failure. We evaluated the radiomodulatory and genotoxic effects of RSV in CHO-k1 and A549 cell lines and in peripheral human blood lymphocytes through both conventional and hypofractionated protocols, due to the widespread use of hypofractionation in recent years. RSV genotoxic and cytotoxic action was assessed at 15 and 60 μM concentrations with the comet and the MTT assay and in cell proliferation experiments. Our results show that RSV administration to tumor cells at a dose of 60 μM exerted a genotoxic effect and that this concentration also had the capacity of modulating the cytomolecular damage induced by 4 and 16 Gy. These doses are delivered in conventional and hypofractionated radiotherapy, respectively. In both treatments, a radiosensitizing effect was evidenced by the decrease in cell viability that was exacerbated over time. These effects were not found in peripheral blood, suggesting that RSV had a dual response. Although the results obtained in CHO-k1 transformed cells corroborated the genotoxic effect of the 60 μM dose of RSV observed in the tumor system, they also showed a radio-protective effect at the lowest dose (15 μM). While more studies are necessary, our results together with the good systemic tolerance of RSV and the lack of toxicity position the compound as a potential candidate for the prevention and treatment of cancer as well as for the optimization of the radiotherapeutic ratio.

Single nucleotide polymorphisms in ATM, TNF-α and IL6 genes and risk of radiotoxicity in breast cancer patients

Although oncological therapies have improved in the last decades, breast cancer (BC) remains a serious health problem worldwide. Radiotherapy (RT) is one of the most frequently used treatments for cancer aimed at eliminating tumor cells. However, it can also alter the surrounding normal tissue, especially the skin, and patient reactions may vary as a result of extrinsic and intrinsic factors. We evaluated the association of gene polymorphisms ATM Asp1853Asn, IL-6 G-174C and TNF-α G-308A involved in central phenotype pathways and development of individual radiosensitivity in BC patients with an exacerbated response to RT. Although univariate analysis results did not show a significant association with this trait, the interaction analysis between polymorphisms showed an increased risk of patients presenting wild-type TNF-α G-308A genotype and mutant IL-6 G-174C genotype, and heterozygous TNF-α G-308A genotype and heterozygous IL-6G-174C genotype. On the other hand, our results showed that breast size and patient age influenced the determination of RT-associated effects. Considering that the trait is multifactorial, other significant elements for the determination of individual radiosensitivity should be considered, together with the establishment of specific polymorphic variants.