RESUMEN
It was suggested that the cryodamage to oocytes' DNA has been responsible for the compromised developmental competence of cryopreserved oocytes. Vitrification of bovine oocytes affected not only cellular components, but also nuclear material. A significant rate of DNA fragmentation was found in bovine frozen or vitrified oocytes analysed by Comet assay regardless of cryopreservation method. Our method of vitrification using droplet system after gentle pre-equilibration treatment is one of the most effective cryopreservation methods employed for bovine oocytes so far, making it possible to develop 30% blastocyst stage embryos. In this study, the extent of DNA damage in bovine oocytes vitrified using three vitrification methods (droplet system, Open Pulled Straw and traditional vitrification in 0.25 ml insemination straws) was compared using Comet assay. Vitrification in droplet system and Open Pull Straws vitrification did not result in detectable cryoinjuries of DNA of bovine oocytes. On the contrary, DNA fragmentation was found in four of 26 oocytes vitrified in 0.25 ml straws (15.4%, p Asunto(s)
Bovinos
, Ensayo Cometa/veterinaria
, Criopreservación/veterinaria
, Daño del ADN
, Oocitos/química
, Animales
, Criopreservación/métodos
, Fragmentación del ADN
, Femenino
, Oocitos/crecimiento & desarrollo
, Oocitos/ultraestructura
RESUMEN
We examined the response to hydrogen peroxide of two L5178Y (LY) sublines which are inversely cross-sensitive to hydrogen peroxide and X-rays: LY-R cells are radio-resistant and hydrogen peroxide-sensitive, whereas LY-S cells are radiosensitive and hydrogen peroxide-resistant. Higher initial DNA breaks and higher iron content (potentially active in the Fenton reaction) were found in the hydrogen peroxide sensitive LY-R cells than in the hydrogen peroxide resistant LY-S cells, whereas the antioxidant defence of LY-R cells was weaker. In particular, catalase activity is twofold higher in LY-S than in LY-R cells. The content of monobromobimane-reactive thiols is 54% higher in LY-S than in LY-R cells. In contrast, the activity of glutathione peroxidase (GPx) is about two times higher in LY-R than in LY-S cells; however, upon induction with selenium the activity increases 15.6-fold in LY-R cells and 50.3-fold in LY-S cells. Altogether, the sensitivity difference is related to the iron content, the amount of the initial DNA damage, as well as to the efficiency of the antioxidant defence system. Differential nuclear translocation of p65-NF-kappaB in LY sublines is due to the more efficient antioxidant defence in LY-S than in LY-R cells.
Asunto(s)
Transporte Activo de Núcleo Celular , Antioxidantes/metabolismo , Núcleo Celular/metabolismo , Hierro/metabolismo , Linfoma/metabolismo , FN-kappa B/metabolismo , Estrés Oxidativo , Animales , Daño del ADN , Relación Dosis-Respuesta a Droga , Humanos , Peróxido de Hidrógeno/farmacología , Modelos Biológicos , Temperatura , Factores de Tiempo , Factor de Transcripción ReIA , Células Tumorales Cultivadas , Rayos XRESUMEN
Hexavalent chromium compounds are well-recognized carcinogens. They easily penetrate the cell membrane and are reduced inside the cell to their trivalent form, which is supposed to react directly with DNA. Chromium is present in some workplaces as well as in water resources and food chain, so it can interact with the mucosa of the gastrointestinal tract. In order to elucidate the genotoxic potency of chromium in human gastric mucosa (GM) cells, the DNA-damaging effect of potassium dichromate (K2Cr2O7) was investigated using alkaline single cell gel electrophoresis (comet assay). Biopsy samples were obtained during gastroscopy from macroscopically healthy tissue of the stomach. Parallel test with human peripheral blood lymphocytes was also performed. Both types of cells were incubated at 37 degrees C with 1.6 mM of K2Cr2O7 for 1 h and after washing, were placed in a chromium-free medium to examine DNA repair. Alkaline single cell gel electrophoresis (comet assay) was used to assess DNA damage and repair. Chromium introduced a damage to DNA both in the GM cells and lymphocytes. The effect induced by K2Cr2O7 in GM cells was comparable with that caused in the lymphocytes. Treated cells were able to recover within a 60-min incubation in a chromium-free medium at 37 degrees C. The results obtained indicate that hexavalent chromium compounds, which may be found in the diet, can interact directly with DNA of the mucosa of the stomach.
Asunto(s)
Mucosa Gástrica/efectos de los fármacos , Linfocitos/efectos de los fármacos , Mutágenos/toxicidad , Dicromato de Potasio/toxicidad , Ensayo Cometa , Daño del ADN , Reparación del ADN , Mucosa Gástrica/metabolismo , Humanos , Técnicas In Vitro , Linfocitos/metabolismoRESUMEN
We have previously found different proportions of iron and copper in nuclei of two sublines of murine lymphoma L5178Y (LY) and proposed a model of chromatin organization with these metal ions at the DNA attachment sites. We now examine the effect of chelators, desferal (DFO, iron-specific) and neocupreine (NEO, copper-specific) on DNA of LY-R and LY-S cells, using the comet and micronuclei frequency tests. There is less copper and more iron in LY-R nuclei than in LY-S nuclei. Accordingly, the effect of NEO is more marked in LY-R than in LY-S cells and in both sublines it is expressed as enhanced tail moment (measure of DNA damage in the comet assay) and increased micronuclei frequency. On the contrary, the effect of DFO on the tail moment is less pronounced in LY-R than in LY-S cells. With increasing DFO concentrations, there is a gradual decrease in the tail moment values below the control level in LY-S cells. In LY-R cells the tail moment values initially increase, then gradually decrease, eventually falling below the control level. This points to a dramatic conformational change that masks the effect of DNA discontinuities. The presence of the latter is indicated by the increase in micronuclei frequency. These results support the postulated differential role of iron and copper ions in maintaining the higher order DNA structure in LY sublines.
Asunto(s)
Quelantes/farmacología , Cobre/metabolismo , ADN de Neoplasias/efectos de los fármacos , Hierro/metabolismo , Animales , Recuento de Células/efectos de los fármacos , Núcleo Celular/química , Núcleo Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Daño del ADN , ADN de Neoplasias/metabolismo , Deferoxamina/farmacología , Electroforesis en Gel de Agar/métodos , Leucemia L5178 , Micronúcleos con Defecto Cromosómico/efectos de los fármacos , Micronúcleos con Defecto Cromosómico/metabolismo , Pruebas de Micronúcleos , Fenantrolinas/farmacología , Células Tumorales Cultivadas/citología , Células Tumorales Cultivadas/efectos de los fármacos , Células Tumorales Cultivadas/metabolismoRESUMEN
In our preceding papers [M. Wojewódzka, M. Kruszewski, T. Iwanenko, A.R. Collins, I. Szumiel, Application of the comet assay for monitoring DNA damage in workers exposed to chronic low dose irradiation: I. Strand breakage, Mutat. Res., 416 (1998) 21-35; M. Kruszewski, M. Wojewódzka, T. Iwanenko, A.R. Collins, I. Szumiel, Application of the comet assay for monitoring DNA damage in workers exposed to chronic low dose irradiation: II. Base damage, Mutat. Res. , 416 (1998) 37-57.], we evaluated the DNA breakage and base damage with the use of comet assay in a group of 49 workers chronically exposed to low doses of ionizing radiation. There was a statistically significant difference in the damage levels between the hazard and control group. In this paper we describe a confounding lack of effect of the smoking habit on the DNA damage in the tested groups. The genotoxic effect of the smoking habit, as well as its modifying effect on genome damage inflicted by other agents, have been firmly established. However, no statistically significant effect of smoking was found in our study, neither in the control nor in the hazard group. This lack of effect was seen in all DNA damage determinations, both direct (DNA strand breakage and alkali-labile lesions) and enzyme-combined (base damage) and did not depend on the comet parameters, which were taken as damage indicators.
Asunto(s)
Daño del ADN , Leucocitos Mononucleares/efectos de los fármacos , Fumar/efectos adversos , ADN/análisis , ADN/efectos de los fármacos , Electroforesis en Gel de Agar/métodos , Endodesoxirribonucleasas , Femenino , Humanos , Masculino , Pruebas de Mutagenicidad/métodos , Fumar/genéticaRESUMEN
Radiosensitive L5178Y-S (LY-S) subline and its parental, more radioresistant L5178Y-R (LY-R) subline differ in DNA double strand break (DSB) rejoining. In this work we examined by comet assay the repair of X-ray-induced DNA damage in LY cells treated with OK-1035, a potent DNA-PK inhibitor. The unirradiated cells differ: the respective tail moment values for LY-R and LY-S cells were 9.62+/-2.84 and 3.52+/-0.1, reflecting the susceptibility to lysis conditions as well as the possible endogenous (oxidative) damage level. The level of initial DNA damage measured after irradiation (8 Gy) at DNA-denaturing pH was the same in both LY sublines: the mean tail moment values +/- SD were 92.93+/-10.39 for LY-R cells and 94.93+/-12.94 for LY-S cells. In LY-S cells the repair of 8 Gy X-ray-induced damage proceeded identically in the presence or absence of 2 mM OK-1035 to the same level of residual damage. In contrast, the level of residual damage in inhibitor treated LY-R cells was considerably higher than that in the untreated cells. Moreover, the inhibitor affected LY-R cells in G1 and S phases and not those in G2, in agreement with cell-cycle specificity of DNA-PK. These results may indicate that the DSB repair defect previously identified in LY-S cells is due to a lack of function of DNA-PK or its impaired activation in the irradiated cells.
Asunto(s)
Reparación del ADN/efectos de los fármacos , Proteínas de Unión al ADN , Hidrazonas/farmacología , Piridonas/farmacología , Animales , Ciclo Celular , Daño del ADN , Reparación del ADN/fisiología , Reparación del ADN/efectos de la radiación , ADN de Neoplasias/efectos de los fármacos , ADN de Neoplasias/metabolismo , ADN de Neoplasias/efectos de la radiación , Proteína Quinasa Activada por ADN , Inhibidores Enzimáticos/farmacología , Leucemia L5178/metabolismo , Ratones , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Proteínas Serina-Treonina Quinasas/metabolismo , Tolerancia a Radiación , Células Tumorales CultivadasRESUMEN
In the preceding paper [M. Wojewodzka, M. Kruszewski, T. Iwanenko, A.R. Collins, I. Szumiel, Application of the comet assay for monitoring DNA damage in workers exposed to chronic low dose irradiation. I. Strand breakage., Mutat. Res. 416 (1998) 21-35], we reported the results of DNA damage examination carried out for a group of people (49 individuals) professionally at risk of exposure to low doses of ionizing radiation as measured by the alkaline comet assay. Here, we used the method in combination with oxidative base damage-specific endonucleases to estimate base damage in the same individuals. These were endonuclease III (endoIII) and formamidopyrimidine glycosylase (FPG). In contrast to the previous investigations, we found no statistically significant difference in base damage between the control and hazard groups. Interestingly, the hazard group exhibited lower level of enzyme-sensitive sites than the control; however, this different was not significant. No correlation of base damage with age was found, similarly as in the case of DNA damage measured by the alkaline comet assay. Interindividual variability of base damage precluded exposure estimation for single individuals, since several members of the control group exhibited high comet parameters.
Asunto(s)
Daño del ADN , ADN/efectos de la radiación , Desoxirribonucleasa (Dímero de Pirimidina) , Proteínas de Escherichia coli , Exposición Profesional , Adulto , Anciano , Estudios de Casos y Controles , ADN/química , ADN/aislamiento & purificación , ADN-Formamidopirimidina Glicosilasa , Endodesoxirribonucleasas , Femenino , Humanos , Leucocitos Mononucleares/química , Leucocitos Mononucleares/efectos de la radiación , Masculino , Persona de Mediana Edad , N-Glicosil Hidrolasas , Monitoreo de Radiación/métodos , Factores de RiesgoRESUMEN
We examined a group of people professionally at risk of exposure to low doses of ionizing radiation (altogether 49 individuals). Age, use of therapeutic drugs, work-related exposure to hazardous agents, previous exposures to diagnostic X-rays, such as patient and nuclear medical examination, were registered. For each individual, the occupational radiation burden received over the past period of 5 years was taken from the official personal records based on film dosimetry controlled every month. A matched group of controls was chosen among the administrative employees (40 individuals). The mean age of the studied population at the time of blood sampling was 49 years (range 24-69). The individuals were divided into groups according to risk of exposure and sex. The alkaline comet assay was used to measure DNA breaks and alkali-labile sites. We compared the mean tail moments, tail length and percentage of DNA in the tail. There was a significant difference between the control and hazard groups in DNA damage. Higher DNA damage was also found for men than for women in the control group. There was no relation of DNA damage to age either in control or hazard group. Additionally, analysis of distributions of tail moment values pointed to a considerable individual diversity even in the control group. Therefore, further investigations were necessary into the suitability of the comet assay as a biological dosimetry method; the results obtained so far warrant such investigations.
Asunto(s)
Daño del ADN , ADN/efectos de la radiación , Exposición Profesional , Adulto , Anciano , Estudios de Casos y Controles , ADN/aislamiento & purificación , Femenino , Dosimetría por Película , Humanos , Leucocitos Mononucleares/efectos de la radiación , Masculino , Persona de Mediana Edad , Energía Nuclear , Monitoreo de Radiación/métodos , Factores de RiesgoRESUMEN
The role of nuclear proteins in protection of DNA against ionizing radiation and their contribution to the radiation sensitivity was examined by an alkaline version of comet assay in two L5178Y (LY) mouse lymphoma cell lines differing in sensitivity to ionizing radiation. LY-S cells are twice more sensitive to ionizing radiation than LY-R cells (D0 values of survival curves are 0.5 Gy and 1 Gy, respectively). Sequential removal of nuclear proteins by extraction with NaCl of different concentrations increased the X-ray induced DNA damage in LY-R nucleoids. In contrast, in the radiation sensitive LY-S cell line, depletion of nuclear proteins practically did not affect DNA damage. Although there is no doubt that the main cause of LYS cells' sensitivity to ionizing radiation is a defect in the repair of double-strand breaks, our data support the concept that nuclear matrix organisation may contribute to the cellular susceptibility to DNA damaging agents.
Asunto(s)
Daño del ADN , Linfoma/patología , Proteínas Nucleares/metabolismo , Tolerancia a Radiación , Animales , Ratones , Células Tumorales Cultivadas , Rayos XRESUMEN
Using a radioresistant subline of murine lymphoma cells L5178(R) DNA damages, their repair and the process of second DNA degradation in dynamics following X-radiation (5 Gy), and in the distant cell progeny have been studied by the method of DNA-comet assay (the alcaline conditions). The repair of DNA damages was found to be finished by 6 h, and by 18 h the second DNA-degradation was observed. The cell progeny was followed throughout 30 generations, with the enhancement of sensitivity to repeat irradiation, and the decrease in repair rate and degree of the second DNA degradation being registered. In the population of the 30th generation, the lack of the second DNA degradation was observed. If the second DNA degradation is indicative of apoptosis, as it is generally considered in literature, it can be supposed that in the distant progeny of irradiated L5178Y(R) cells radiation induces no programmed cell death and is not a trigger of apoptosis.
Asunto(s)
Apoptosis/efectos de la radiación , Daño del ADN , Reparación del ADN , Leucemia L5178/genética , Leucemia L5178/patología , Animales , Daño del ADN/efectos de la radiación , Reparación del ADN/efectos de la radiación , Ratones , Tolerancia a Radiación , Factores de Tiempo , Células Tumorales CultivadasRESUMEN
Two sublines of L5178Y (LY) murine lymphoma, differing in sensitivity to hydrogen peroxide, served as a cellular model for examination of the antioxidant defense system. The contribution of catalase, glutathione peroxidase (G-Px) and glutathione were evaluated. Sensitivity to 3-amino-1,2,4-triazole (AMT), inhibitor of catalase, was higher in LY-R (hydrogen peroxide sensitive) than in LY-S (hydrogen peroxide resistant) cells. Accordingly, activity of catalase was twofold lower in LY-R than in LY-S cells. G-Px activity was about two times higher in LY-R than in LY-S cells. After induction with selenium it increased 15.6 times in LY-R cells and 50.3 times in LY-S cells. Reduced glutathione (GSH) content (and possibly other monobromobimane-reactive thiols) were determined fluorimetrically with monobromobimane and fluorescence found 54% higher in LY-S than in LY-R cells. Inhibition of catalase caused GSH decrease in LY-S cells; this decrease was abrogated by inducing G-Px by selenium treatment. On the contrary, in LY-R cells inhibition of catalase decreased GSH content only slightly and selenium treatment did not further change the GSH level. DNA damage (estimated by "comet" assay) was the same in hydrogen peroxide-treated cells in the presence or absence of AMT; however, after induction of G-Px by selenium, DNA damage was considerably lowered. This sparing effect of selenium was accompanied by decreased growth inhibition in selenium pretreated, hydrogen peroxide-treated cell cultures.
Asunto(s)
Antioxidantes/metabolismo , Peróxido de Hidrógeno/farmacología , Leucemia L5178/metabolismo , Amitrol (Herbicida)/farmacología , Animales , Catalasa/antagonistas & inhibidores , Catalasa/metabolismo , Daño del ADN , Resistencia a Medicamentos , Inducción Enzimática/efectos de los fármacos , Inhibidores Enzimáticos/farmacología , Radicales Libres/metabolismo , Glutatión/metabolismo , Glutatión Peroxidasa/biosíntesis , Glutatión Peroxidasa/metabolismo , Ratones , Selenio/farmacología , Células Tumorales CultivadasRESUMEN
The L5178Y (LY) murine lymphoma subline, LY-R, is more radioresistant and more sensitive to camptothecin (CPT, inhibitor of topisomerase I) than the second subline used in our investigation, LY-S. Post-irradiation treatment with 3 microM CPT enhanced the radiosensitivity of LY-S cells (D0 decrease from 0.52 to 0.34 Gy), but did not change it in LY-R cells. Treatment with 2 mM benzamide [BZ, inhibitor of poly (ADP-ribosylation)] before x-rays and CPT increased the radiosensitivity of LY-R cells (D0 decrease from 1.15 to 0.52) without further modification of radiosensitivity of LY-S cells. Activity of topoisomerase I was diminished 10 min after x-irradiation (5 Gy) in LY-S, but not in LY-R cells. The data on DNA damage (fluorescent halo or comet assays) showed that the ultimate fate of the cells did not depend on the DNA damage pattern estimated immediately after treatment (e.g. the damage was greater in x-rays plus CPT than in BZ plus x-rays plus CPT treated LY-R cells, although the radiosensitivity was less). Aphidicolin (inhibitor of DNA polymerases alpha and delta) applied concomitantly with CPT in cells not pretreated with BZ prevented the increase in DNA damage in LY-R cells, but was without effect in LY-S cells. Taking into account the differential inhibition by x-rays of DNA synthesis in LY sublines and its reversion by BZ in LY-S but not in LY-R cells, we conclude that the pattern of DNA damage observed by the methods applied depended on the status of DNA replication.
Asunto(s)
Antineoplásicos Fitogénicos/farmacología , Benzamidas/farmacología , Camptotecina/farmacología , Daño del ADN , Tolerancia a Radiación , Inhibidores de Topoisomerasa I , Animales , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/efectos de la radiación , Replicación del ADN , Leucemia L5178/patología , Leucemia L5178/terapia , Ratones , Poli Adenosina Difosfato Ribosa/metabolismo , Células Tumorales Cultivadas , Rayos XRESUMEN
Cells from the L5178Y murine lymphoma subline LY-R are twice as resistant to killing by ionizing radiation than the subline LY-S. In contrast, LY-R cells are more sensitive to killing by H2O2, the effect being more pronounced at 37 degrees C than 0 degree C. Initial DNA damage after H2O2 treatment (both temperatures, 5 min) has been estimated by the 'comet' assay (single-cell gel electrophoresis) and fluorescent halo technique. According to both methods, the initial damage is significantly higher in LY-R cells, particularly that inflicted at 0 degree C. Differences between DNA unwinding and rewinding abilities at pH 9 and 6.9 (estimated by the fluorescent halo technique) point to a considerable difference in pH-9-labile damage between the sublines, as observed previously for x-irradiated cells (Kapiszewska et al. 1992). In contrast to findings with x-irradiated cells, however, after H2O2 treatment this damage is more extensive in LY-R cells than in LY-S cells. Thus, the initial pH-9-labile damage corresponds to the pattern of sensitivity to H2O2 and x-rays. We suggest that this is caused by different proportions of cuprous and ferric ions found in the nuclei of LY sublines and by the different ability of these ions to react with H2O2 and water radiolysis products. The copper/iron ratio in the nucleus is 1.31 in LY-R cells and 4.84 in LY-S cells.