RESUMEN
Cadmium (Cd) is an essential material used in the battery, metal-coating, and alloy industries. In addition to these industrial uses, it is also a component of cigarette smoke. Therefore, exposure to cadmium is widespread and presents a considerable health concern. Cadmium is known to be a carcinogen; however, the possible mechanism of carcinogenesis with regards to the activation and inactivation of cancer-related genes has not yet been fully elucidated. In this study, amplification, expression, and point mutation of cancer-related genes associated with Cd-induced cell transformation in BALB/c-3T3 cells were studied. Six proto-oncogenes (K-ras, c-myc, c-fos, c-jun, c-sis, and erbB), as well as the p53 tumor suppressor, were investigated for gene amplification using differential polymerase chain reaction (PCR), while the expression of the proteins produced by these genes was evaluated by Western blot analysis. Point mutations in K-ras and p53 were studied by PCR restriction fragment length polymorphism analysis and DNA sequencing. There were no point mutations observed in codons 12, 13, and 61 of K-ras or in exons 4-10 of p53 and no observed differences in the levels of any of the proteins studied. Among 10 Cd-induced transformed cell lines, significant gene amplification was found for c-myc and c-jun in 50% and 80% of the cell lines, respectively. Chromosome painting was performed to confirm that this amplification was not simply due to additional copies of the chromosomes carrying these oncogenes. In addition, reverse-transcription PCR (RT-PCR) was performed to confirm increased expression of c-myc and c-jun. These results suggest that cell transformation induced by Cd may be attributed, at least in part, to gene amplification of c-myc and c-jun and that some of the Cd-transformed cells may possess neoplastic potential resulting from genomic instability.
Asunto(s)
Cadmio/toxicidad , Transformación Celular Neoplásica/efectos de los fármacos , Expresión Génica/efectos de los fármacos , Proteínas Proto-Oncogénicas/biosíntesis , Proteínas Proto-Oncogénicas/genética , Animales , Western Blotting , Pintura Cromosómica , ADN/biosíntesis , ADN/aislamiento & purificación , Cartilla de ADN , Electroforesis en Gel de Agar , Electroforesis en Gel de Poliacrilamida , Genes jun/efectos de los fármacos , Genes myc/efectos de los fármacos , Ratones , Ratones Endogámicos BALB C , Mutación , Polimorfismo de Longitud del Fragmento de Restricción , ARN/biosíntesis , ARN/aislamiento & purificación , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Células Tumorales CultivadasRESUMEN
As the measurement of chromosomal translocations increases in popularity for quantifying prior radiation exposure, information on the possible decline of these "stable" aberrations over time is urgently needed. We report here information about the persistence of radiation-induced chromosome aberrations in vivo over the life span of a rodent. Female C57BL/6 mice were given a single whole-body acute exposure of 0, 1, 2, 3 or 4 Gy (137)Cs gamma rays at 8 weeks of age. Chromosome aberrations were analyzed from peripheral blood samples at various intervals between 1 day and 21 months after exposure. Aberrations were detected by painting chromosomes 2 and 8. Translocations decreased dramatically during the first 3 months after irradiation, beyond which time the frequencies remained relatively constant out to 1 year, when the effects of aging and clonal expansion became significant. Both reciprocal and nonreciprocal translocations increased with age in the unexposed control animals and were involved in clones. As expected of unstable aberrations, dicentrics decreased rapidly after exposure and reached baseline levels within 3 months. These results indicate that the persistence of translocations induced by ionizing radiation is complicated by aging and clonal expansion and that these factors must be considered when quantifying translocations at long times after exposure. These results have implications for biological dosimetry in human populations.
Asunto(s)
Aberraciones Cromosómicas , Cromosomas/efectos de la radiación , Monitoreo del Ambiente/métodos , Rayos gamma , Linfocitos/efectos de la radiación , Envejecimiento/genética , Animales , Radioisótopos de Cesio , Pintura Cromosómica , Células Clonales/efectos de la radiación , Femenino , Linfocitos/patología , Ratones , Ratones Endogámicos C57BL , Dosis de Radiación , Translocación Genética , Irradiación Corporal TotalRESUMEN
In this paper, we present data on chromosome aberration frequencies in mice which served as unexposed controls in a variety of radiation and chemical toxicology experiments conducted in our laboratory in recent years. All chromosome aberration data were obtained by chromosome painting. In peripheral blood lymphocytes from 102 animals, the frequencies of translocations and insertions increased significantly with age. No increase with age was seen for dicentrics or acentric fragments. When the data were analyzed by strain, the age-related increase in translocation frequencies was observed only in the 71 homozygous C57BL/6 mice and not in any of the three heterozygous strains. Very few aberrations of any type were observed in 62 bone marrow samples, and no effect of age was seen for any aberration type in this tissue. These results are similar to those observed in unexposed humans, and suggest that the increase in translocations is not the result of accumulated damage from chronic 'background' environmental exposures but instead may be due to biological processes associated with aging.
Asunto(s)
Envejecimiento/genética , Aberraciones Cromosómicas , Animales , Heterocigoto , Ratones , Ratones Endogámicos C57BLRESUMEN
Fluorescence in situ hybridization, or chromosome painting, has become an invaluable tool in the cytogenetic evaluation of historical or chronic exposure because it can be used to detect stable genetic damage, such as translocations, which persist through cell division, quickly and easily. The recent development of chromosome-specific composite DNA probes for the mouse has allowed the use of chromosome painting in this commonly used animal model. In order to measure the persistence of radiation-induced translocations, C57BL/6 female mice were given a whole body acute dose of 0, 1, 2, 3 or 4 Gy 137Cs gamma rays at 8 weeks of age. Metaphase chromosomes from both peripheral blood and bone marrow cells were obtained from four mice in each dose group at 1, 8, 15 and 30 days post-irradiation. Chromosomes 2 and 8 were painted, while the remaining chromosomes were counterstained with propidium iodide. DAPI counterstain was used to differentiate between translocations and dicentrics because it brightly labels the centromeric heterochromatin. The equivalent of 100 cells from each tissue was scored from each mouse. The results show that the percentage of reciprocal translocations, at least at doses of 3 Gy or lower, did not decrease with time in either tissue. In contrast, the frequency of non-reciprocal translocations induced by doses of 3 Gy or lower, remained unchanged in the peripheral blood, but decreased after a week in the bone marrow, then remained constant. An increase in these two types of aberration was observed between 15 and 30 days in the bone marrow and may have been due to clonal expansion. Dicentrics decreased with time in both tissues, almost none remained in the bone marrow after 8 days. These data suggest that reciprocal translocations are persistent and will serve as an effective biodosimeter for radiation exposure.