RESUMEN
The potential health effects of inhaling carbon nanotubes are important because of possible exposures in occupational settings. Previously, we have shown mice that have inhaled multiwalled carbon nanotubes have suppressed systemic immune function. Here, we show the mechanisms for this immune suppression. Mice were exposed to 0, 0.3 or 1 mg m(-3) multiwalled carbon nanotubes for 6 h per day for 14 consecutive days in whole-body inhalation chambers. Only those exposed to a dose of 1 mg m(-3) presented suppressed immune function; this involved activation of cyclooxygenase enzymes in the spleen in response to a signal from the lungs. Spleen cells from exposed animals partially recovered their immune function when treated with ibuprofen, a drug that blocks the formation of cyclooxygenase enzymes. Knockout mice without cyclooxygenase enzymes were not affected when exposed to multiwalled carbon nanotubes, further confirming the importance of this enzyme in suppression. Proteins from the lungs of exposed mice suppressed the immune function of spleen cells from normal mice, but not those from knockout mice. Our findings suggest that signals from the lung can activate signals in the spleen to suppress the immune function of exposed mice.
Asunto(s)
Sistema Inmunológico/efectos de los fármacos , Nanotubos de Carbono/toxicidad , Animales , Líquido del Lavado Bronquioalveolar/química , Líquido del Lavado Bronquioalveolar/inmunología , Proliferación Celular/efectos de los fármacos , Ciclooxigenasa 2/deficiencia , Ciclooxigenasa 2/genética , Ciclooxigenasa 2/metabolismo , Inhibidores de la Ciclooxigenasa/farmacología , Modelos Animales de Enfermedad , Ibuprofeno/farmacología , Exposición por Inhalación/efectos adversos , Pulmón/metabolismo , Activación de Linfocitos/efectos de los fármacos , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Tamaño de la Partícula , Bazo/citología , Bazo/inmunología , Linfocitos T/efectos de los fármacos , Linfocitos T/inmunología , Factor de Crecimiento Transformador beta/metabolismoRESUMEN
We have recently reported that 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) inhibits epidermal growth factor (EGF) withdrawal-induced apoptosis in the human mammary epithelial cell line MCF-10A. We hypothesized that TCDD-mediated inhibition of apoptosis was due to its ability to stimulate the EGF receptor (EGFR) pathway. Indeed, in the present studies, the EGFR inhibitor AG1478 was able to prevent TCDD-, EGF-, and transforming growth factor alpha (TGF-alpha)-dependent cell recovery and inhibition of apoptosis. These effects were specific for an EGFR-mediated pathway because cotreatment with AG825, an erbB2 inhibitor, had little effect on apoptosis. In addition, TCDD was able to mimic the EGF and TGF-alpha signaling as demonstrated by increasing Akt and extracellular signal-regulated kinase 1,2 phosphorylation. These effects were dependent on EGFR activity because AG1478, but not AG825, was able to prevent EGF-, TGF-alpha, or TCDD-mediated Akt and extracellular signal-regulated kinase 1,2 phosphorylation. The ability of TCDD to stimulate the EGFR pathway and inhibit apoptosis may be due to the ability of TCDD to increase expression of TGF-alpha, a ligand for EGFR. Treatment with 10 nM TCDD increased TGF-alpha mRNA at 2 h and TGF-alpha protein at 6 h. These data suggest a mechanism whereby TCDD is able to inhibit apoptosis in human mammary epithelial cells by stimulating TGF-alpha production, resulting in an autocrine effect.
Asunto(s)
Apoptosis/efectos de los fármacos , Dibenzodioxinas Policloradas/farmacología , Proteínas Serina-Treonina Quinasas , Factor de Crecimiento Transformador alfa/biosíntesis , Apoptosis/fisiología , Benzotiazoles , Mama/citología , Mama/efectos de los fármacos , Mama/metabolismo , Recuento de Células , Activación Enzimática , Inhibidores Enzimáticos/farmacología , Receptores ErbB/antagonistas & inhibidores , Receptores ErbB/fisiología , Humanos , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteína Quinasa 3 Activada por Mitógenos , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Fosforilación , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Proto-Oncogénicas c-akt , Quinazolinas , ARN Mensajero/biosíntesis , ARN Mensajero/genética , Factor de Crecimiento Transformador alfa/genética , Tirfostinos/farmacologíaRESUMEN
Flow cytometry offers numerous advantages over traditional techniques for measuring intracellular Ca(2+) in lymphoid and nonlymphoid cells. In particular, the heterogeneity of cell responses can be defined by flow cytometry, and multiparameter analyses permit the determination of intracellular Ca(2+) in surface-marker-defined target cells as well as correlation of changes in Ca(2+) with other biochemical markers, including ligand binding. This article presents several established methods for measuring intracellular Ca(2+) by flow cytometry in lymphoid and nonlymphoid cells. Examples are provided for determination of Ca(2+) in human peripheral blood leukocytes and two human epithelial cell lines grown in monolayer. In addition, applications are reviewed or presented for correlating changes in intracellular Ca(2+) with other cell parameters, including cell cycle analysis, changes in cell membrane integrity, and the induction of apoptosis markers. Finally, a number of novel sample handling capabilities useful for performing kinetic analyses of Ca(2+) changes by flow cytometry are now available and one application is presented which is finding utility in pharmacologic studies.
Asunto(s)
Calcio/análisis , Calcio/metabolismo , Citometría de Flujo/métodos , Líquido Intracelular/química , Líquido Intracelular/metabolismo , Compuestos de Anilina/metabolismo , Benzofuranos/metabolismo , Calibración , Adhesión Celular , Línea Celular , Membrana Celular/metabolismo , Células Cultivadas , Quelantes/metabolismo , Células Epiteliales/citología , Células Epiteliales/metabolismo , Citometría de Flujo/instrumentación , Colorantes Fluorescentes/metabolismo , Humanos , Imidazoles/metabolismo , Leucocitos Mononucleares/citología , Leucocitos Mononucleares/metabolismo , Propidio/metabolismo , Receptores de Formil Péptido , Receptores Inmunológicos/genética , Receptores Inmunológicos/metabolismo , Receptores de Péptidos/genética , Receptores de Péptidos/metabolismo , Espectrometría de Fluorescencia , Transfección , Xantenos/metabolismoRESUMEN
Previous studies have demonstrated that 2,3,7, 8-tetrachlorodibenzo-p-dioxin (TCDD) increases cell recovery in the human mammary epithelial cell line MCF-10A grown under growth factor-restricted conditions. TCDD was also found to mimic growth factor signaling pathways by stimulating the tyrosine phosphorylation of numerous effector molecules, and increased phosphatidylinositol 3-kinase (PI3K) activity in the absence of exogenously added growth factors. In the present studies, we have expanded on these initial results to show that TCDD (3-30 nM) increases cell recovery on days 2-6 by as much as 80% when insulin or epidermal growth factor (EGF) was removed from the media. The mechanism for this effect appears to be complex as TCDD inhibited apoptosis stimulated by EGF, or EGF and insulin, withdrawal by almost 80% as determined by Annexin V binding. However, withdrawal of insulin alone did not induce apoptosis even though TCDD did increase cell number in its absence. These results were corroborated by immunoblot analysis of poly(ADP-ribose) polymerase cleavage. Since TCDD stimulates PI3K activity, the phosphorylation status of Akt, a serine/threonine kinase that mediates PI3K-dependent inhibition of apoptosis, was examined. Immunoblot analysis revealed that TCDD causes a transient increase in the phosphorylated form of Akt that peaks at 6 h and disappears by 12 h. It appears that EGF stimulates an anti-apoptotic pathway, while insulin signals a pro-mitogenic pathway. By stimulating or mimicking one or both of these pathways TCDD may alter tightly regulated growth pathways in the MCF-10A cell line.
Asunto(s)
Apoptosis/efectos de los fármacos , Mama/efectos de los fármacos , Factor de Crecimiento Epidérmico/farmacología , Insulina/farmacología , Dibenzodioxinas Policloradas/farmacología , Mama/citología , Humanos , Hidrólisis , Proteína Oncogénica v-akt , Fosforilación , Poli(ADP-Ribosa) Polimerasas/metabolismo , Proteínas Oncogénicas de Retroviridae/metabolismo , Células Tumorales CultivadasRESUMEN
Flow cytometry is an emerging technology that has numerous applications to immunotoxicity testing. The use and development of high-speed single-cell laser-based assays capable of quantitation of fluorescence, light scatter, and electrical impedance measurements can provide important information on xenobiotic-induced toxicity in defined target cell populations. The purpose of this article is to briefly review established and emerging immunotoxicology assays that use flow cytometry. In the coming years it is likely that many new flow cytometry assays will be developed and validated that will improve the sensitivity and perhaps specificity of immunotoxicity testing. Since flow cytometry is readily adaptable to high-throughput screening, it is also likely that this technology will increasingly find its place in the preclinical testing of drugs and chemicals in the pharmaceutical and chemical industries.
Asunto(s)
Citometría de Flujo/métodos , Técnicas Inmunológicas , Toxicología/métodos , Animales , Apoptosis , Biomarcadores , Calcio/metabolismo , Ciclo Celular , Supervivencia Celular , Daño del ADN , Citometría de Flujo/tendencias , Colorantes Fluorescentes , Humanos , Luz , Activación de Linfocitos , Dispersión de Radiación , Toxicología/tendenciasRESUMEN
Carcinogenic polycyclic aromatic hydrocarbons and a halogenated aromatic hydrocarbon, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), were evaluated for their effects on intracellular Ca2+ in the human mammary epithelial cell line MCF-10A. After two 18-h incubations with MCF-10A cells, benzo[a]pyrene (BaP; 1, 3, and 10 microM) produced a dose-dependent increase in intracellular Ca2+. 7,12-Dimethylbenz[a]anthracene increased Ca2+ at 10 microM, whereas 3-methylcholanthrene and TCDD did not. The Ca2+-elevating effect of BaP appeared to be dependent on the influx of extracellular Ca2+, as addition of the Ca2+ chelator EGTA to the extracellular medium prevented the increase in Ca2+. MCF-10A cells were found by polymerase chain reaction to express cytochrome P4501A and P4501B isozymes as well as the aryl hydrocarbon receptor and aryl hydrocarbon receptor nuclear translocator mRNAs associated with cytochrome P450 induction. Certain cytochrome P450-derived metabolites, including benzo[a]pyrene-7,8-diol (BP-diol) and benzo[a]pyrene-7,8-diol-9,10-epoxide (BPDE), were more effective in increasing Ca2+ than was BaP. The Ca2+-elevating effect of BP-diol was prevented by alpha-naphthoflavone, a cytochrome P4501A and P4501B inhibitor, but not by the antioxidant N-acetylcysteine. These results suggest that cytochrome P450-dependent formation of BPDE from BP-diol is a major mechanism required for elevation of Ca2+ in MCF-10A cells.
Asunto(s)
Mama/efectos de los fármacos , Calcio/metabolismo , Carcinógenos/farmacología , Proteínas de Unión al ADN , Células Epiteliales/efectos de los fármacos , Hidrocarburos Policíclicos Aromáticos/farmacología , 7,8-Dihidro-7,8-dihidroxibenzo(a)pireno 9,10-óxido/farmacología , 9,10-Dimetil-1,2-benzantraceno/farmacología , Acetilcisteína/farmacología , Translocador Nuclear del Receptor de Aril Hidrocarburo , Benzoflavonas/farmacología , Benzopirenos/farmacología , Mama/citología , Mama/metabolismo , Línea Celular , Supervivencia Celular/efectos de los fármacos , Inhibidores Enzimáticos del Citocromo P-450 , Sistema Enzimático del Citocromo P-450/genética , Sistema Enzimático del Citocromo P-450/metabolismo , Dihidroxidihidrobenzopirenos/antagonistas & inhibidores , Dihidroxidihidrobenzopirenos/farmacología , Ácido Egtácico/farmacología , Células Epiteliales/metabolismo , Humanos , Metilcolantreno/farmacología , Dibenzodioxinas Policloradas/farmacología , Hidrocarburos Policíclicos Aromáticos/antagonistas & inhibidores , ARN Mensajero/metabolismo , Receptores de Hidrocarburo de Aril/genética , Factores de Tiempo , Factores de Transcripción/genéticaRESUMEN
It has been well established that certain polycyclic aromatic hydrocarbons (PAHs), such as 7,12-dimethylbenz[a]anthracene (DMBA), 3-methylcholanthrene (3MC), and benzo[a]pyrene (BaP), produce immunotoxicity and cancer in rodents and that these effects are also likely seen in humans. Our laboratory has found that polycyclic aromatic hydrocarbons (PAHs) produce an increase in intracellular Ca2+ in lymphocytes that appears to correlate with their immunotoxicity. Specifically, immunotoxic PAHs, such as DMBA and BaP, have been shown to produce a sustained increase in intracellular Ca2+ in lymphocytes, whereas nonimmunosuppressive PAHs, such as benzo[e]pyrene (BeP) and anthracene, do not. Our studies previously demonstrated that the rapid increase in intracellular Ca2+ produced by DMBA in HPB-ALL T cells was caused by protein tyrosine kinase (PTK) activation in human T cells, leading to tyrosine phosphorylation of phospholipase C (PLCgamma) and IP3-dependent Ca2+ mobilization. However, the specificity of PTK activation by PAHs was not established. In the present studies, we extend our observations of PTK activation by examining a number of PAHs for their effects on total and specific (Fyn and ZAP-70) PTK activity. We show that 10 microM concentrations of PAHs nonspecifically and rapidly (within 5 min) stimulate PTKs in the HPB-ALL human T cell line. BeP and anthracene were found to be nearly as effective at increasing total tyrosine kinase activity as DMBA, 3MC, and BaP, observed 5 min after exposure. We found that only immunotoxic PAHs activated the Fyn and ZAP-70 PTKs at 10 min, but total PTK activity was still increased by nonimmunotoxic PAHs, BeP, or anthracene after 10 min of exposure. These studies demonstrate that immunotoxic PAHs increase total and specific PTK activity in the human HPB-ALL T cell line. Thus the rapid increase in PTK activity produced by PAHs may not correlate with the immunotoxicity of these agents.
Asunto(s)
Carcinógenos Ambientales/farmacología , Proteína Tirosina Quinasa p56(lck) Específica de Linfocito/efectos de los fármacos , Linfocitos/efectos de los fármacos , Hidrocarburos Policíclicos Aromáticos/farmacología , 9,10-Dimetil-1,2-benzantraceno/farmacología , Análisis de Varianza , Antracenos/farmacología , Benzo(a)pireno/farmacología , Benzopirenos/farmacología , Línea Celular , Humanos , Proteína Tirosina Quinasa p56(lck) Específica de Linfocito/metabolismo , Linfocitos/enzimología , Relación Estructura-ActividadRESUMEN
Previous studies in this laboratory have shown that polycyclic aromatic hydrocarbons (PAHs) alter Ca2+ homeostasis and inhibit activation of both B and T lymphocytes obtained from rodents and humans. In the present studies, we demonstrate that alpha-naphthoflavone (ANF), an inhibitor of cytochrome P4501A activity, reduced the Ca2+ elevation produced by BaP in human peripheral blood mononuclear cell (HPBMC) lymphocytes. These results suggested that BaP metabolites may play a role in intracellular Ca2+ homeostasis in human lymphocytes. Reactive oxidative intermediates of BaP produced in HPMBC are known to be highly carcinogenic and have also been shown to be immunosuppressive. We examined the effects of benzo(a)pyrene (BaP), 7,12-dimethylbenz(a)anthracene (DMBA), benzo(e)pyrene (BeP), and anthracene, as well as certain BaP metabolites, on the levels of intracellular Ca2+ and glutathione in HPBMC. While BaP, DMBA, BeP, and anthracene did not cause a statistically significant decrease in GSH in HPBMC at concentrations of 1 or 10 microM following a 6-, 48-, or 72-hr exposure, reactive BaP metabolites including 4,5-epoxide BaP and 7,8-diol-9,10-epoxide BaP consistently produced a 20-30% depletion of glutathione in HPBMC following a 6-hr treatment period. These BaP metabolites also elevated intracellular Ca2+ in HPBMC during a 6-hr incubation. Results of these experiments suggest that metabolism of BaP to certain epoxide metabolites may be responsible for sulfhydryl damage leading to transient GSH depletion and Ca2+ elevation. These results are consistent with the hypothesis that sulfhydryl damage by certain PAH metabolites may lead to altered Ca2+ homeostasis, leading to inhibition of cell activation and proliferation in HPBMC.