RESUMEN
We report here the complete genome sequence of a WU polyomavirus (WUPyV) isolate, also known as human polyomavirus 4, collected in 2016 from a patient in Arkansas with an acute respiratory infection. Isolate hPyV4/USA/AR001/2016 has a double-stranded DNA genome of 5,229 bp in length.
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Using target capture of viral nucleic acid and next-generation sequencing, we generated the complete genomes of two novel human parainfluenza virus 1 isolates. Isolates AR001 (accession no. KX570602) and NM001 (accession no. KX639498) were collected 3 months apart from pediatric patients with acute respiratory infection from Arkansas and New Mexico, respectively.
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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.
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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
Polycyclic aromatic hydrocarbons are environmental pollutants known to be carcinogenic and immunotoxic. In intact cell assays, benzo[a]pyrene (B[a]P) disrupts Ca(2+) homeostasis in both immune and nonimmune cells, but the molecular mechanism is undefined. In this study, B[a]P and five metabolites are examined for their ability to alter Ca(2+) transport across microsomal membranes. Using a well-defined model system, junctional SR vesicles from skeletal muscle, we show that a single o-quinone metabolite of B[a]P, B[a]P-7,8-dione, can account for altered Ca(2+) transport across microsomal membranes. B[a]P-7,8-dione induces net Ca(2+) release from actively loaded vesicles in a dose-, time-, and Ca(2+)-dependent manner. In the presence of 5 microM extravesicular Ca(2+), B[a]P-7,8-dione exhibited threshold and EC(50) values of 0.4 and 2 microM, respectively, and a maximal release rate of 2 micromol of Ca(2+) min(-1) mg(-1). The mechanism by which B[a]P-7,8-dione enhanced Ca(2+) efflux was further investigated by measuring macroscopic fluxes and single RyR1 channels reconstituted in bilayer lipid membranes and direct measurements of SERCA catalytic activity. B[a]P-7,8-dione (< or = 20 microM) had no measurable effect on initial rates of Ca(2+) accumulation in the presence of ruthenium red to block ryanodine receptor (RyR1), nor did it alter Ca(2+)-dependent (thapsigargin-sensitive) ATPase activity. B[a]P-7,8-dione selectively altered the function of RyR1 in a time-dependent diphasic manner, first activating then inhibiting channel activity. Considering that RyR1 and its two alternate isoforms are broadly expressed in mammalian cells and their important role in Ca(2+)-signaling, the present results reveal a mechanism by which metabolic bioactivation of B[a]P may mediate RyR dysfunction of pathophysiological significance.
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Benzo(a)pireno/farmacología , Benzopirenos/farmacología , Calcio/metabolismo , Microsomas/efectos de los fármacos , Canal Liberador de Calcio Receptor de Rianodina/fisiología , Animales , Transporte Biológico/efectos de los fármacos , ATPasas Transportadoras de Calcio/metabolismo , Técnicas In Vitro , Microsomas/enzimología , Microsomas/metabolismo , Oxidación-Reducción , Quinonas/metabolismo , Conejos , Retículo Sarcoplasmático/efectos de los fármacos , Retículo Sarcoplasmático/enzimología , Retículo Sarcoplasmático/metabolismo , ATPasas Transportadoras de Calcio del Retículo SarcoplásmicoRESUMEN
The article highlighted in this issue is "An Aryl Hydrocarbon Receptor Independent Mechanism of JP-8 Jet Fuel Immunotoxicity in Ah-Responsive and Ah-Nonresponsive Mice" by Andrew C. Dudley, Margie M. Peden-Adams, Jackie EuDaly, Richard S. Pollenz, and Deborah E. Keil (pp. 251-259).
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Genómica , Proteoma/análisis , Toxicología/métodos , Animales , ADN/análisis , Perfilación de la Expresión Génica , Humanos , Ratones , Ratones Noqueados , Ratones Transgénicos , Análisis de Secuencia por Matrices de Oligonucleótidos , Proteoma/genética , Receptores de Hidrocarburo de Aril/genéticaRESUMEN
During the past decade there has been significant progress made in understanding how environmental agents, drugs, certain chemicals present in the diet, and occupational agents affect the immune system of animals and humans. Polycyclic aromatic hydrocarbons (PAHs) are an important class of environmentally prevalent xenobiotics that exert complex effects on the immune system. These agents, typified by benzo(a)pyrene (BaP), have been shown to alter antigen and mitogen receptor signaling pathways, leading to suppression of humoral and cell-mediated immunity, and at high exposure levels to activation of genes involved in apoptosis in lymphoid cells. Interestingly, at low exposure levels, PAHs may actually augment cell signaling pathways, resulting in immune enhancement or an adjuvant effect. While the biochemical targets and mechanisms responsible for immune modulation are still under investigation, several themes are evolving. PAHs, principally through their cytochrome-P450-derived metabolites, activate oxidative and electrophilic signaling pathways in lymphoid and nonlymphoid cells, including myeloid, epithelial, and other cells. Although PAHs affect signaling pathways in nonlymphoid cells leading to complex interactions between antigen-specific and nonspecific immune and inflammatory responses, this brief review focuses on the mechanisms of signaling by environmentally prevalent PAHs in human lymphocytes. Understanding the mechanisms by which xenobiotics alter adaptive and nonadaptive immune responses may shed light on the etiology of environmental and occupational immune diseases.
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Contaminantes Ambientales/análisis , Linfocitos/inmunología , Hidrocarburos Policíclicos Aromáticos/análisis , Animales , Contaminantes Ambientales/toxicidad , Humanos , Linfocitos/citología , Hidrocarburos Policíclicos Aromáticos/toxicidad , Transducción de SeñalRESUMEN
Polycyclic aromatic hydrocarbons affect cells in many ways, including covalent modifications of DNA, participation in redox cycling, and alterations in cellular signaling pathways. Similarly, exposure to ultraviolet (UV) light may modify DNA, generate reactive oxygen species, and alter signaling. Because environmental conditions may interact to affect cellular functions, we investigated the combined effects of benzo[a]pyrene (BaP) and UV light in a cell line in which BaP-induced alterations in Ca(2+) homeostasis have previously been shown. Exposure of MCF-10A cells to BaP (18 h) followed by a brief (5 min) exposure to UVA resulted in resistance to trypsinization of cells grown on type I collagen (Vitrogen). This effect was not seen following treatment with BaP or UVA alone nor with benzo(e)pyrene (BeP)+UVA. BaP+UVA light also caused actin filaments to reorganize from typical stress fibers to substrate-associated aggregates of actin and caused depletion of cellular adenosine triphosphate (ATP). The effects of BaP+UVA on adhesion and actin aggregate formation were partially prevented by treatment with reduced glutathione. Depletion of cellular ATP affected resistance to trypsinization and actin organization in a similar manner. Thus, these studies suggest a redox-sensitive interaction between BaP+UVA light to deplete cellular ATP levels, resulting in resistance to trypsinization and actin filament reorganization in MCF-10A cells.
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Adenosina Trifosfato/metabolismo , Benzo(a)pireno/farmacología , Citoesqueleto/efectos de los fármacos , Tripsina/farmacología , Rayos Ultravioleta , Actinas/efectos de los fármacos , Actinas/metabolismo , Actinas/efectos de la radiación , Antimicina A/farmacología , Adhesión Celular/efectos de los fármacos , Adhesión Celular/efectos de la radiación , Recuento de Células , Línea Celular , Citoesqueleto/metabolismo , Citoesqueleto/efectos de la radiación , Desoxiglucosa/farmacología , Relación Dosis-Respuesta a Droga , Resistencia a Medicamentos , Ácido Egtácico/farmacología , Glutatión/farmacología , Humanos , Estrés OxidativoRESUMEN
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.
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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.
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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.
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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.
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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/genéticaAsunto(s)
Contaminantes Ambientales/inmunología , Hipersensibilidad/etiología , Sistema Inmunológico/efectos de los fármacos , Contaminantes Atmosféricos/inmunología , Alergia e Inmunología/tendencias , Humanos , Contaminantes del Suelo/inmunología , Toxicología/tendencias , Contaminantes Químicos del Agua/inmunologíaRESUMEN
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.
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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
Numerous studies have demonstrated an association between polycyclic aromatic hydrocarbons (PAHs) and lymphocyte toxicity. The present study shows that, consistent with its effects on Ca2+ homeostasis, benzo[a]pyrene (BaP) induces apoptosis in Daudi cells. Terminal deoxynucleotidal transferase-mediated dUTP-biotin nick end labeling (TUNEL) analysis at 18 h revealed a significant increase in the number of cells undergoing apoptosis in response to BaP (75%), BaP-7, 8-dihydrodiol (110%), and BaP-7,8-9,10-diol epoxide (BPDE) (215%) over DMSO vehicle control cultures. By 36 h, the trend toward increasing numbers of apoptotic cells continued with the parent compound producing a 125% increase over control values and the 7, 8-dihydrodiol and BPDE metabolites producing 195% and 370% increases over controls, respectively. DNA fragmentation assays demonstrated the presence of internucleosomal cleavage products consistent with the increasing numbers of TUNEL-positive cells responding to PAHs at 18 and 36 h. Analysis of poly(ADP-ribose) polymerase (PARP) protein in BaP- and BaP-7,8-dihydrodiol-treated cells strongly suggested the involvement of cysteine proteases by the appearance of an 85-kD fragment derived from hydrolytic cleavage of PARP, a phenomenon that has been associated with apoptosis in many systems. Immunoblot analysis demonstrated that both BaP and its 7,8-dihydrodiol metabolite affected a pathway involving Bcl-2 and Bax cytosolic proteins. Daudi cells undergoing apoptosis at 36 h in response to 10 microM BaP, the parent compound, expressed moderately reduced amounts of Bcl-2 (78% of vehicle controls). At the same time point, the 7,8-dihydrodiol and BDPE metabolites at 3 microM resulted in Bcl-2 protein expression that was 52% of that seen in vehicle controls. Parallel samples analyzed for expression of Bax protein displayed a 130% increase over vehicle control in Bax expression in response to the parent compound, while the 7,8-dihydrodiol metabolite produced a 257% increase in Bax. Furthermore, the effects on increased Bax expression were observed as early as 3 h after PAH exposure. The apoptotic response to PAHs in Daudi cells was sensitive to 4-h pretreatment with 0.3 microM alpha-naphthoflavone (ANF), a known inhibitor of cytochrome P450. In TUNEL assays of cells exposed to PAHs following pretreatment with ANF, at 18 h there was a significant reduction in the number of cells undergoing apoptosis in response to ANF compared to cells that were not pretreated with the compound. The effect of the parent compound at 18 h was completely blocked with ANF pretreatment, while ANF exerted a relatively weaker, but significant, effect on BaP-7, 8-dihydrodiol-induced apoptosis. With regard to modulation of expression of apoptosis-related proteins, Bax expression was restored to that observed in vehicle-control cultures at all time points tested (3, 18, and 36 h). Bcl-2 expression was most responsive to ANF at later time points following PAH exposure (18 and 36 h); however, Bcl-2 appeared to be more sensitive to the effects of ANF alone. Taken together, these data suggest that modulation of Bcl-2 family proteins, perhaps secondary to altered Ca2+ homeostasis, plays an important role in human B cell apoptosis induced by BaP.
Asunto(s)
Apoptosis/efectos de los fármacos , Linfocitos B/efectos de los fármacos , Benzo(a)pireno/toxicidad , Dihidroxidihidrobenzopirenos/toxicidad , 7,8-Dihidro-7,8-dihidroxibenzo(a)pireno 9,10-óxido/farmacología , Factor Natriurético Atrial/farmacología , Línea Celular , Fragmentación del ADN , Humanos , Poli(ADP-Ribosa) Polimerasa-1 , Poli(ADP-Ribosa) Polimerasas , Proteínas/metabolismo , Proteínas Proto-Oncogénicas/biosíntesis , Proteínas Proto-Oncogénicas c-bcl-2/biosíntesis , Proteína X Asociada a bcl-2RESUMEN
Previous studies in this laboratory have shown that polycyclic aromatic hydrocarbons, such as benzo[a]pyrene (BaP), and certain halogenated aromatic hydrocarbons, such as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), modulate receptor signaling pathways in human lymphoid and non-lymphoid cells. We have recently demonstrated that BaP produces a weak mitogenic signal in human mammary epithelial cells, perhaps by mimicking growth factor signaling pathways. In the present studies we found that BaP and TCDD activated insulin-like growth factor (IGF-I) signaling pathways under insulin-deficient conditions. The effects of BaP and TCDD were evaluated in the human MCF-10A mammary epithelial cell line grown under epidermal growth factor- and insulin-dependent conditions. BaP (0.3 microM) and TCDD (30 nM) were found to restore a moderate insulin-like signal in MCF-10A cells grown in the absence of added insulin. TCDD was more potent and produced better activation of cell growth than did BaP. Both TCDD and BaP appeared to mimic signaling through the IGF-I receptor (IGF-IR), as evidenced by increased tyrosine phosphophorylation of IGF-IRbeta, IRS-1 and Shc. In addition, both BaP and TCDD significantly increased the activity of phosphatidylinositol 3-kinase (PI3K). The PI3K inhibitor LY294002 was found to inhibit the growth-promoting effects of TCDD seen under insulin-deficient conditions. The results of these studies show that under certain conditions BaP and TCDD can mimic growth factor signaling pathways in human mammary epithelial cells, demonstrating that environmentally prevalent carcinogenic compounds may alter cell growth in human mammary epithelial cells via mimicry of growth factor receptor signaling pathways.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales , Proteínas Adaptadoras del Transporte Vesicular , Benzo(a)pireno/farmacología , Mama/efectos de los fármacos , Mama/fisiología , Carcinógenos/farmacología , Factor I del Crecimiento Similar a la Insulina/fisiología , Dibenzodioxinas Policloradas/farmacología , Transducción de Señal/efectos de los fármacos , Tirosina/metabolismo , Mama/metabolismo , División Celular/efectos de los fármacos , Línea Celular , Supervivencia Celular/efectos de los fármacos , Medios de Cultivo , Activación Enzimática/efectos de los fármacos , Células Epiteliales/efectos de los fármacos , Células Epiteliales/metabolismo , Células Epiteliales/fisiología , Humanos , Insulina/deficiencia , Fosfatidilinositol 3-Quinasas/metabolismo , Fosforilación/efectos de los fármacos , Proteínas/metabolismo , Receptor IGF Tipo 1/metabolismo , Proteínas Adaptadoras de la Señalización Shc , Transducción de Señal/fisiología , Proteína Transformadora 1 que Contiene Dominios de Homología 2 de SrcRESUMEN
Previous studies performed in this laboratory have shown that certain benzo(a)pyrene (BaP) metabolites, such as benzo(a)pyrene-7,8-dihydrodiol (BaP-7,8-diol) and benzo(a)pyrene-7,8-dihydrodiol-9,10-epoxide (BPDE), were more effective in elevating intracellular Ca2+ in normal human peripheral blood mononuclear cell (HPBMC) T and B cells than was BaP. Additionally, it has been shown that the suppression of human T cell mitogenesis produced by polycyclic aromatic hydrocarbons (PAHs) and certain BaP metabolites is reversed by treatment with alpha-naphthoflavone (ANF), a cytochrome P450 1A and 1B inhibitor. ANF also diminishes the elevation in intracellular calcium (Ca2+) produced by BaP in HPBMC. In the present studies, we further defined the relationships between intracellular Ca2+ elevation produced by BaP and two immunotoxic P450-derived metabolites, BaP-7,8-diol and BPDE in the Daudi human B cell line. At 1, 4, and 18 h, both BaP-7,8-diol and BPDE produced a significant rise in intracellular Ca2+. This effect, however, was not observed with BaP or benzo(e)pyrene (BeP), a nonimmunotoxic PAH. To evaluate the potential role of cytochrome P450 metabolism in PAH-induced Ca2+ elevation, Daudi cells were pretreated with ANF for 4 h, followed by treatment with BaP metabolites for 18 h. ANF completely reversed the rise in Ca2+ produced by BaP-7,8-diol, but had no effect on the Ca2+ elevation produced by BPDE. These results suggest that BPDE may be the ultimate P450 metabolite responsible for Ca2+ elevation in human B cells. BaP-7,8-diol and BPDE were found to increase tyrosine phosphorylation in Daudi whole cell lysates and to increase tyrosine phosphorylation of two important Src-related protein tyrosine kinases (PTKs), Lyn and Syk. Inhibition of tyrosine phosphorylation by herbimycin A was found to largely prevent the increase in intracellular Ca2+ produced by BaP-7,8-diol and BPDE, suggesting that Ca2+ elevation is coupled to increased tyrosine phosphorylation in Daudi. BPDE was found to produce a statistically significant increase in tyrosine phosphorylation of Lyn and Syk within 10 min of exposure. Collectively, these data demonstrate that certain P450-derived metabolites of BaP may be responsible for PTK activation and an increase intracellular Ca2+, which may alter antigen receptor signaling in human B cells.
Asunto(s)
Linfocitos B/efectos de los fármacos , Benzo(a)pireno/toxicidad , Calcio/metabolismo , Sistema Enzimático del Citocromo P-450/metabolismo , Proteínas Tirosina Quinasas/metabolismo , 7,8-Dihidro-7,8-dihidroxibenzo(a)pireno 9,10-óxido/toxicidad , Linfocitos B/metabolismo , Benzo(a)pireno/metabolismo , Benzoflavonas/farmacología , Benzoquinonas , Línea Celular , Inhibidores Enzimáticos del Citocromo P-450 , Dihidroxidihidrobenzopirenos/toxicidad , Humanos , Lactamas Macrocíclicas , Proteínas Tirosina Quinasas/antagonistas & inhibidores , Quinonas/farmacología , Rifabutina/análogos & derivadosRESUMEN
Previous studies have demonstrated that polycyclic aromatic hydrocarbons (PAHs), such as benzo[a]pyrene (BaP) and 7,12-dimethybenz[a]anthracene (DMBA), and possibly 2,3,7,8-tetrachlorodibenzo(p)dioxin (TCDD), may exert their immunosuppressive effects by altering intracellular Ca2+ homeostasis in lymphocytes. In these studies, we examined the effects of two immunosuppressive PAHs (BaP and DMBA), two nonimmunosuppressive PAHs (benzo[e]pyrene (BeP) and anthracene (ANTH)), and TCDD on intracellular Ca2+ levels in surface marker-defined human peripheral blood mononuclear cells (HPBMC). BaP and DMBA, but not BeP and ANTH, were found to produce a time-dependent increase in intracellular Ca2+ with maximal effects achieved following 42- to 66-hr exposures. In a series of studies with HPBMC obtained from 10 donors exposed in vitro for 42 hr, BaP and DMBA were found to produce a significant increase in Ca2+ in CD3+ T cells, CD19+ B cells, and CD14+ monocytes. BeP and ANTH did not produce a statistically significant increase in Ca2+ in the group of donors, but occasionally produced an apparent nonspecific elevation of Ca2+ in HPBMC from individual donors. Interestingly, TCDD produced a small and statistically significant increase in Ca2+ only in B cells analyzed for the pooled 10 donors. Certain BaP metabolites, such as the 7,8-dihydrodiol and the 7,8-diol-9,10-epoxide, were more effective in elevating Ca2+ in HPBMC lymphocytes at 20 hr than was BaP. These results demonstrate in normal HPBMC that immunosuppressive PAHs alter intracellular Ca2+ homeostasis in B cells, T cells, and monocytes, and suggest that P450 metabolism may play an important role in the immunotoxicity of certain PAHs.
Asunto(s)
Antígenos CD/análisis , Calcio/metabolismo , Líquido Intracelular/efectos de los fármacos , Líquido Intracelular/metabolismo , Leucocitos Mononucleares/efectos de los fármacos , Leucocitos Mononucleares/metabolismo , Hidrocarburos Policíclicos Aromáticos/toxicidad , 9,10-Dimetil-1,2-benzantraceno/sangre , 9,10-Dimetil-1,2-benzantraceno/toxicidad , Adulto , Subgrupos de Linfocitos B/efectos de los fármacos , Subgrupos de Linfocitos B/metabolismo , Benzo(a)pireno/metabolismo , Benzo(a)pireno/toxicidad , Calcio/sangre , Humanos , Inmunosupresores/toxicidad , Hidrocarburos Policíclicos Aromáticos/sangre , Subgrupos de Linfocitos T/efectos de los fármacos , Subgrupos de Linfocitos T/metabolismo , Factores de TiempoRESUMEN
Flow cytometry is a unique technology useful in the examination of effects of immunotoxic agents on target cells of the immune system. The purpose of this workshop was to provide an overview of the use of flow cytometry in new and established models of immunotoxicity, with emphasis on the potential applications, assay validation, and potential pitfalls. This overview begins with a discussion of methods useful in the assessment of Ca2+-dependent mechanisms of lymphoid cell activation in surface marker-defined human B cells, T cells, and monocytes. A discussion of the use of flow cytometry in analysis of apoptosis is also presented in this paper. The second paper presents data on the development and use of flow cytometry as an alternative to a Cr51 release assay for an assessment of cytotoxic T cell activation. The use of surface markers for characterizing and distinguishing the effects of chemical irritants from sensitizers is next presented, followed by an overview of the use of fluorescent probes to assess cell thiol status and overall oxidant-induced injury to lymphoid cells. Finally, an interlaboratory study designed to compare and evaluate the use of flow cytometry procedures in rat splenic cell subtyping is presented. Overall, these studies demonstrate the utility of flow cytometry assays in immunotoxicologic research, but further efforts are needed in the validation of many of these assays for routine use in immunotoxicologic testing.
Asunto(s)
Citometría de Flujo , Sistema Inmunológico/efectos de los fármacos , Inmunotoxinas/toxicidad , Alérgenos/toxicidad , Animales , Ciclo Celular/efectos de los fármacos , Ciclo Celular/inmunología , Ciclo Celular/fisiología , Tamaño de la Célula/efectos de los fármacos , Humanos , Sistema Inmunológico/citología , Irritantes/toxicidad , Activación de Linfocitos/efectos de los fármacos , Activación de Linfocitos/inmunología , Linfocitos/efectos de los fármacos , Linfocitos/fisiología , Estrés Oxidativo , Linfocitos T Citotóxicos/efectos de los fármacos , Linfocitos T Citotóxicos/fisiologíaRESUMEN
Previous studies have shown that polycyclic aromatic hydrocarbons (PAHs) mobilize intracellular Ca2+ in human T cells by inositol trisphosphate-dependent mechanisms resulting from activation of phospholipase C-gamma by SRC-related protein tyrosine kinases, thereby mimicking antigen-receptor activation. Ca2+ appears to play an important second messenger role in growth factor control of cell proliferation in human mammary epithelial cells (HMEC), such as the epidermal growth factor receptor pathway. The purpose of the present studies was to determine if PAHs are able to increase intracellular Ca2+ in primary cultures of HMEC and increase cell proliferation. Two carcinogenic and two non-carcinogenic PAHs were tested for their ability to increase intracellular Ca2+ in HMEC. The carcinogenic PAHs dimethylbenz[a]anthracene (DMBA) and benzo[a] pyrene (BaP) were able to cause Ca2+ elevation in HMEC at early time points (2 h) and caused sustained alterations in Ca2+ homeostasis (18 h). DMBA showed maximal effects at early time points (2 h), while BaP showed maximal effects on sustained Ca2+ (18 h). 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD), a potent dioxin and tumor promoter, produced maximal Ca2+ elevation at 2 h, with a return to near baseline levels by 6 h. The non-carcinogenic PAHs benzo[e]pyrene and anthracene did not significantly alter intracellular Ca2+ at any time point. alpha-Naphthoflavone significantly reduced the Ca2+ response induced by BaP treatment, but not by DMBA or TCDD, suggesting that P450 1A or 1B metabolism of BaP may be important in the sustained Ca2+ elevating response. In evaluating the effects of BaP on HMEC proliferation, BaP was found to increase the number of cells recovered after 4 days in culture in the absence or presence of various concentrations of epidermal growth factor. These studies provide initial evidence that Ca2+ signaling may be associated with mitogenesis in HMEC, which may play a role in tumor promotion and progression produced by PAHs.