RESUMEN
The pink-eyed unstable mutation, p(un), is the result of a 70 kb tandem duplication within the murine pink-eyed, p, gene. Deletion of one copy of the duplicated region by homologous deletion/recombination occurs spontaneously in embryos and results in pigmented spots in the fur and eye. Such deletion events are inducible by a variety of DNA damaging agents, as we have observed previously with both fur- and eye-spot assays. Here we describe a study of the effect of exposure to benzo[a]pyrene (B[a]P) at different times of development on reversion induction in the eye. Previously we, among others, have reported that the retinal pigment epithelium (RPE) displays a position effect variegation phenotype in the pattern of pink-eyed unstable reversions. Following an acute exposure to B[a]P or X-rays on the tenth day of gestation an increased frequency of reversion events was detected in a distinct region of the adult RPE. Examining exposure at different times of eye development reveals that both B[a]P and X-rays result in an increased frequency of reversion events, though the increase was only significant following B[a]P exposure, similar to our previous report limited to exposure on the tenth day of gestation. Examination of B[a]P-exposed RPE in the present study revealed distinct regions where the induced events lie and that the positions of these regions are found at increasing distances from the optic nerve the later the time of exposure. This position effect directly reflects the previously observed developmental pattern of the RPE, namely that cells in the regions most distal from the optic nerve are proliferating most vigorously. The numbers and positions of RPE cells displaying the transformed (pigmented) phenotype strongly advocate the proposal that dividing cells are at highest risk to deletions induced by carcinogens.
Asunto(s)
Benzo(a)pireno/farmacología , Color del Ojo/efectos de los fármacos , Epitelio Pigmentado Ocular/efectos de los fármacos , Recombinación Genética/efectos de los fármacos , Animales , Animales Recién Nacidos , Benzo(a)pireno/toxicidad , Carcinógenos , División Celular/efectos de los fármacos , Color del Ojo/genética , Eliminación de Gen , Ratones , Ratones Endogámicos C57BL , Neoplasias/etiología , Neoplasias/genética , Nervio Óptico/metabolismo , Fenotipo , Epitelio Pigmentado Ocular/metabolismo , Factores de Tiempo , Rayos XRESUMEN
Multiple genetic changes are required for the development of a malignant tumor cell and many environmentally induced cancers show a delayed onset of > 20 years following exposure. In fact, the frequency of genetic changes in cancer cells is higher than can be explained by random mutation. A high level of genetic instability in a subpopulation of cells may be caused by a mutator phenotype transmitted through many cell divisions. We have determined the effects of irradiation of parental male mice on the frequency and characteristics of mitotically occurring DNA deletion events at the p(un) locus in the offspring. Reversion of the p(un) marker in mouse embryos is due to deletion of 70 kb of DNA resulting in fur spots in the offspring. We found that irradiation of male mice caused a significantly higher frequency of large spots in the offspring, indicative of the induction of DNA deletions early in embryo development. These deletion events occurred, however, many cell divisions after irradiation. The present data indicate that exposure of the germline to ionizing radiation results in induction of delayed DNA deletions in offspring mice.
Asunto(s)
Mutación , Rayos X , Animales , Masculino , Ratones , Efectos de la RadiaciónRESUMEN
We have generated mice deficient in O6-methylguanine DNA methyltransferase activity encoded by the murine Mgmt gene using homologous recombination to delete the region encoding the Mgmt active site cysteine. Tissues from Mgmt null mice displayed very low O6-methylguanine DNA methyltransferase activity, suggesting that Mgmt constitutes the major, if not the only, O6-methylguanine DNA methyltransferase. Primary mouse embryo fibroblasts and bone marrow cells from Mgmt -/- mice were significantly more sensitive to the toxic effects of the chemotherapeutic alkylating agents 1,3-bis(2-chloroethyl)-1-nitrosourea, streptozotocin and temozolomide than those from Mgmt wild-type mice. As expected, Mgmt-deficient fibroblasts and bone marrow cells were not sensitive to UV light or to the crosslinking agent mitomycin C. In addition, the 50% lethal doses for Mgmt -/- mice were 2- to 10-fold lower than those for Mgmt +/+ mice for 1,3-bis(2chloroethyl)-1-nitrosourea, N-methyl-N-nitrosourea and streptozotocin; similar 50% lethal doses were observed for mitomycin C. Necropsies of both wild-type and Mgmt -/mice following drug treatment revealed histological evidence of significant ablation of hematopoietic tissues, but such ablation occurred at much lower doses for the Mgmt -/- mice. These results demonstrate the critical importance of O6-methylguanine DNA methyltransferase in protecting cells and animals against the toxic effects of alkylating agents used for cancer chemotherapy.
Asunto(s)
Alquilantes/toxicidad , O(6)-Metilguanina-ADN Metiltransferasa/genética , Animales , Antibióticos Antineoplásicos/farmacología , Antineoplásicos Alquilantes/farmacología , Carcinógenos/farmacología , Carmustina/farmacología , Dacarbazina/análogos & derivados , Dacarbazina/farmacología , Genotipo , Sistema Hematopoyético/anatomía & histología , Hígado/enzimología , Metilnitronitrosoguanidina/farmacología , Ratones , Ratones Noqueados , Mitomicina/farmacología , Modelos Biológicos , Estreptozocina/farmacología , TemozolomidaRESUMEN
Deletions and other genome rearrangements are associated with carcinogenesis and inheritable diseases. The pink-eyed unstable (pun) mutation in the mouse is caused by duplication of a 70-kb internal fragment of the p gene. Spontaneous reversion events in homozygous pun/pun mice occur through deletion of a duplicated sequence. Reversion events in premelanocytes in the mouse embryo detected as black spots on the gray fur of the offspring were inducible by the carcinogen x-rays, ethyl methanesulfonate, methyl methanesulfonate, ethyl nitrosourea, benzo[a]pyrene, trichloroethylene, benzene, and sodium arsenate. The latter three carcinogens are not detectable with several in vitro or in vivo mutagenesis assays. We studied the molecular mechanism of the carcinogen-induced reversion events by cDNA analysis using reverse transcriptase-PCR method and identified the induced reversion events as deletions. DNA deletion assays may be sensitive indicators for carcinogen exposure.
Asunto(s)
Carcinógenos/farmacología , Proteínas Portadoras , Proteínas de la Membrana/genética , Mutagénesis , Pruebas de Mutagenicidad/métodos , Animales , Color del Cabello/genética , Ratones , Ratones Endogámicos C57BL , Recombinación Genética/efectos de los fármacos , Eliminación de SecuenciaRESUMEN
Deletions and other genome rearrangements can be caused by radiation and are associated with carcinogenesis and inheritable diseases. The pink-eyed unstable (p(un)) mutation in the mouse is caused by a gene duplication and reverts to wild type by deletion of one copy. Reversion events in the mouse embryo were detected as black spots on the fur of the animals or microscopically as partially black hair in a background of colorless hair. The frequency of partially black hair was increased by x-rays at very low doses. A linear dose-response relation was found between 1 and 100 centigray.
Asunto(s)
Eliminación de Gen , Mutación/efectos de la radiación , Animales , Relación Dosis-Respuesta en la Radiación , Embrión de Mamíferos/efectos de la radiación , Femenino , Color del Cabello/genética , Color del Cabello/efectos de la radiación , Masculino , Exposición Materna , Melanocitos/efectos de la radiación , Ratones , Ratones Endogámicos C57BL , Ratones Mutantes , Familia de Multigenes , Pruebas de MutagenicidadRESUMEN
The Saccharomyces cerevisiae DEL assay detects a wide variety of nonmutagenic carcinogens (Schiestl et al. (1989) Carcinogenesis, 10, 1445-1455). This study shows the effect on DEL recombination of 8 carcinogenic compounds (o-toluidine, hexamethylphosphoramide, safrole, acrylonitrile, benzene, diethylhexylphthalate, phenobarbital and diethylstilbestrol) and 2 noncarcinogenic compounds (caprolactam and benzoin). These chemicals have been selected by the Program on Chemical Safety for the evaluation of short-term tests for carcinogens, because sufficient carcinogenicity data for these compounds exist, and because they are difficult to detect with the Salmonella assay. 5 of 8 carcinogens reproducibly gave a strong positive response and the noncarcinogen benzoin was negative. Thus, 60% of the chemicals tested in this study have been correctly identified with the DEL assay compared to only 20% with the Salmonella assay.