RESUMO
Hexavalent chromium (Cr(VI)) compounds are known human lung carcinogens. Solubility plays an important role in its carcinogenicity with the particulate or insoluble form being the most potent. Of the particulate Cr(VI) compounds, zinc chromate appears to be the most potent carcinogen; however, very few studies have investigated its carcinogenic mechanism. In this study, we investigated the ability of chronic exposure to zinc chromate to induce numerical chromosome instability. We found no increase in aneuploidy after a 24 h exposure to zinc chromate, but with more chronic exposures, zinc chromate induced concentration- and time-dependent increases in aneuploidy in the form of hypodiploidy, hyperdiploidy, and tetraploidy. Zinc chromate also induced centrosome amplification in a concentration- and time-dependent manner in both interphase and mitotic cells after chronic exposure, producing cells with centriolar defects. Furthermore, chronic exposure to zinc chromate induced concentration- and time-dependent increases in spindle assembly checkpoint bypass with increases in centromere spreading, premature centromere division, and premature anaphase. Last, we found that chronic exposure to zinc chromate induced a G2 arrest. All together, these data indicate that zinc chromate can induce chromosome instability after prolonged exposures.
Assuntos
Centrossomo/efeitos dos fármacos , Cromatos/toxicidade , Instabilidade Cromossômica/efeitos dos fármacos , Fibroblastos/efeitos dos fármacos , Pulmão/efeitos dos fármacos , Fuso Acromático/efeitos dos fármacos , Compostos de Zinco/toxicidade , Aneuploidia , Linhagem Celular , Humanos , Pulmão/citologia , Tamanho da Partícula , SolubilidadeRESUMO
Hexavalent chromium Cr(VI) is a respiratory toxicant and carcinogen, with solubility playing an important role in its carcinogenic potential. Zinc chromate, a water insoluble or 'particulate' Cr(VI) compound, has been shown to be carcinogenic in epidemiology studies and to induce tumors in experimental animals, but its genotoxicity is poorly understood. Our study shows that zinc chromate induced concentration-dependent increases in cytotoxicity, chromosome damage and DNA double strand breaks in human lung cells. In response to zinc chromate-induced breaks, MRE11 expression was increased and ATM and ATR were phosphorylated, indicating that the DNA double strand break repair system was initiated in the cells. In addition, our data show that zinc chromate-induced double strand breaks were only observed in the G2/M phase population, with no significant amount of double strand breaks observed in G1 and S phase cells. These data will aid in understanding the mechanisms of zinc chromate toxicity and carcinogenesis.
Assuntos
Cromatos/toxicidade , Instabilidade Cromossômica , Quebras de DNA de Cadeia Dupla , DNA/efeitos dos fármacos , Fibroblastos/efeitos dos fármacos , Pulmão/efeitos dos fármacos , Mutagênicos/toxicidade , Compostos de Zinco/toxicidade , Proteínas Mutadas de Ataxia Telangiectasia , Ciclo Celular/efeitos dos fármacos , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , DNA/metabolismo , Proteínas de Ligação a DNA/metabolismo , Relação Dose-Resposta a Droga , Fibroblastos/metabolismo , Fibroblastos/patologia , Humanos , Pulmão/metabolismo , Pulmão/patologia , Proteína Homóloga a MRE11 , Fosforilação , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Regulação para CimaRESUMO
Defective B-lymphopoiesis has been associated with development of auto-antibodies and auto-immunity in a number of autoimmune-prone strains of mice. The flaky skin (fsn) mutation results in development of chronic inflammation and auto-immunity. Associated with the development of auto-immunity is the hyperactivation of B-lymphocytes and production of auto-antibodies. We, therefore, undertook a detailed examination of B-lineage precursors in the bone marrow of fsn/fsn mice. We observed a rapid age-related loss of the pre-B and immature B cells. It was also noted that an accumulation of early precursor populations occurs coincident with the loss of Fr.D and Fr.E bone marrow B cell populations indicating a developmental block or accumulation of pro-B cells in 7 and 10 week old fsn/fsn mice. Our data suggests changes in the fsn/fsn bone-marrow microenvironment that results in senescence of B cell development.
Assuntos
Envelhecimento/imunologia , Doenças Autoimunes/genética , Linfócitos B/imunologia , Células-Tronco Hematopoéticas/imunologia , Depleção Linfocítica , Linfopoese/genética , Fatores Etários , Envelhecimento/genética , Animais , Doenças Autoimunes/patologia , Linfócitos B/patologia , Células da Medula Óssea/imunologia , Células da Medula Óssea/patologia , Diferenciação Celular/genética , Diferenciação Celular/imunologia , Células Cultivadas , Células-Tronco Hematopoéticas/patologia , Cinética , Linfopoese/imunologia , CamundongosRESUMO
Hexavalent chromium (Cr(VI)) is a widespread environmental contaminant and a known human carcinogen, generally causing bronchial cancer. Recent studies have shown that the particulate forms of Cr(VI) are the potent carcinogens. Particulate Cr(VI) is known to induce a spectrum of DNA damage such as DNA single strand breaks, Cr-DNA adducts, DNA-protein crosslinks and chromosomal aberrations. However, particulate Cr(VI)-induced DNA double strand breaks (DSBs) have not been reported. Thus, the aim of this study was to determine if particulate Cr(VI)-induces DSBs in human bronchial cells. Using the single cell gel electrophoresis assay (comet assay), showed that lead chromate-induced concentration dependent increases in DSBs with 0.1, 0.5, 1 and 5 microg/cm2 lead chromate inducing a 20, 50, 67 and 109% relative increase in the tail integrated intensity ratio, respectively. Sodium chromate at concentrations of 1, 2.5 and 5 microM induced 38, 78 and 107% relative increase in the tail integrated intensity ratio, respectively. We also show that genotoxic concentrations of lead chromate activate the ataxia telangiectasia mutated (ATM) protein, which is thought to play a central role in the early stages of DSB detection and controls cellular responses to this damage. The H2A.X protein becomes rapidly phosphorylated on residue serine 139 in cells when DSBs are introduced into the DNA by ionizing radiation. By using immunofluorescence, we found that lead chromate-induced concentration-dependent increases in phosphorylated H2A.X (r-H2A.X) foci formation with 0.1, 0.5, 1, 5 and 10 microg/cm2 lead chromate inducing a relative increase in the number of cells with r-H2A.X foci formation of 43, 51, 115 and 129%, respectively.
Assuntos
Cromatos/toxicidade , Dano ao DNA , Chumbo/toxicidade , Pulmão/citologia , Pulmão/efeitos dos fármacos , Proteínas Mutadas de Ataxia Telangiectasia , Proteínas de Ciclo Celular/metabolismo , Ensaio Cometa , Proteínas de Ligação a DNA/metabolismo , Relação Dose-Resposta a Droga , Fibroblastos , Imunofluorescência , Histonas/metabolismo , Humanos , Fosforilação , Proteínas Serina-Treonina Quinases/metabolismo , Compostos de Sódio/toxicidade , Proteínas Supressoras de Tumor/metabolismo , Regulação para Cima/efeitos dos fármacosRESUMO
Mice homozygous for the flaky skin (fsn) single gene mutation have a severe hyperproliferative disease resulting in a complex phenotype, which includes widespread inflammation and autoimmunity. This study sought to characterize lymphocyte function of flaky skin mutant mice. Flaky skin lymphocytes show enhanced proliferation with in vitro mitogen stimulated spleen cells, as well as enriched splenic B- and T-cells. The production of IL-4 by fsn/fsn T-lymphocytes is increased dramatically compared with normal controls. Flaky skin lymphocytes exhibited increased responsiveness to IL-2, IL-4 and IL-7 in the absence of pre-activation, enhanced IgE production in response to ovalbumin immunization, and constitutive STAT6 activation. These data indicate that the cytokines IL-2, IL-4 and IL-7 likely contribute to the lymphocyte activation in fsn/fsn mutant mice. This lymphocyte hyperactivation may result in the development of systemic autoimmunity in fsn/fsn mutant mice.
Assuntos
Linfócitos B/metabolismo , Proliferação de Células , Interleucina-4/metabolismo , Ativação Linfocitária/fisiologia , Linfócitos T/metabolismo , Animais , Autoimunidade , Interleucina-2/metabolismo , Interleucina-7/metabolismo , Camundongos , Camundongos Mutantes , Fenótipo , Fator de Transcrição STAT6 , Baço , Transativadores/metabolismoRESUMO
Mice homozygous.for the flaky skin (fsn) single gene mutation have a severe hyperproliferative disease resulting complex phenotype, which includes widespread inflammation and autoimmunity. Flaky skin mice have several serological and pathological features that share similarities with the human systemic autoimmune disease systemic lupus erythematosus (SLE). Analyses of the antinuclear and anti-dsDNA autoantibodies in fsn/fsn mice indicate that they are low titer IgG antibodies. These low titer anti-dsDNA autoantibodies can ultimately form immune complex deposition in the glomeruli associated kidney damage. IgE antibodies were identified in the immune complex deposition, however their role in the pathology is not determined. It is hypothesized that the mechanism of autoantibody production and autoimmune disease pathogenesis in mice homozygous for the fsn mutation is initiated by non-specific polyclonal activation of B-lymphocytes resulting in the synthesis of low affinity autoantibodies.
Assuntos
Anticorpos Antinucleares/biossíntese , Psoríase/imunologia , Animais , Anticorpos Antinucleares/sangue , DNA/imunologia , Imunofluorescência , Imunoglobulina E/análise , Interleucina-4/fisiologia , Camundongos , Camundongos Mutantes , Psoríase/genéticaRESUMO
Similar to murine models with compromised CD22/SHP-1 function, flaky skin (fsn) mutant mice exhibit lymphocyte hyperactivation and an autoimmune phenotype characterized by circulating autoantibodies to dsDNA and glomerulonephritis. Immunophenotyping of fsn/fsn splenic B cells was performed to determine if abnormalities in CD22 expression contributed to the phenotype. We identified an expansion of an IgM(bright) CD22lo population consistent with immature B-lymphocytes. While normal B-lymphocytes require IL-4 to achieve down-modulation of CD22 expression in response to BCR cross-linking, culture with anti-IgM alone led to reduced CD22 expression in fsn/fsn mice. Furthermore, when IL-4 was added to fsn/fsn cultures, no further reduction in CD22 expression was observed. This suggested that fsn/fsn B cells were pre-activated in vivo by chronic IL-4 exposure. A portion of these CD22lo cells expressed the B-1 surface marker CD11b. We contend that decreased activation thresholds among CD22lo B-lymphocytes contributes to the expansion of immature and B-1 B cell populations and to the development of autoimmune pathology in fsn/fsn mice.