Clark Lantz
Work Summary
We are interested in the effects of early life exposures to environmental toxicants on lung growth and development. We determine if the early life exposures leads to adult disease.
We are interested in the effects of early life exposures to environmental toxicants on lung growth and development. We determine if the early life exposures leads to adult disease.
Paracrine ATP signaling in the lung epithelium participates in a variety of innate immune functions, including mucociliary clearance, bactericide production, and as an initiating signal in wound repair. We evaluated the effects of chronic low-dose arsenic relevant to U.S. drinking water standards (i.e., 10 ppb [130nM]) on airway epithelial cells. Immortalized human bronchial epithelial cells (16HBE14o-) were exposed to 0, 130, or 330nM arsenic (as Na-arsenite) for 4-5 weeks and examined for wound repair efficiency and ATP-mediated Ca(2+) signaling. We found that chronic arsenic exposure at these low doses slows wound repair and reduces ATP-mediated Ca(2+) signaling. We further show that arsenic compromises ATP-mediated Ca(2+) signaling by altering both Ca(2+) release from intracellular stores (via metabotropic P2Y receptors) and Ca(2+) influx mechanisms (via ionotropic P2X receptors). To better model the effects of arsenic on ATP-mediated Ca(2+) signaling under conditions of natural exposure, we cultured tracheal epithelial cells obtained from mice exposed to control or 50 ppb Na-arsenite supplemented drinking water for 4 weeks. Tracheal epithelial cells from arsenic-exposed mice displayed reduced ATP-mediated Ca(2+) signaling dynamics similar to our in vitro chronic exposure. Our findings demonstrate that chronic arsenic exposure at levels that are commonly found in drinking water (i.e., 10-50 ppb) alters cellular mechanisms critical to airway innate immunity.
PMID: 21684831;PMCID: PMC3230447;Abstract:
Background: Chronic arsenic exposure is a worldwide health problem. How arsenic exposure promotes a variety of diseases is poorly understood, and specific relationships between experimental and human exposures are not established. We propose phenotypic anchoring as a means to unify experimental observations and disease outcomes. Objectives: We examined the use of phenotypic anchors to translate experimental data to human pathology and investigated research needs for which phenotypic anchors need to be developed. Methods: During a workshop, we discussed experimental systems investigating arsenic dose/exposure and phenotypic expression relationships and human disease responses to chronic arsenic exposure and identified knowledge gaps. In a literature review, we identified areas where data exist to support phenotypic anchoring of experimental results to pathologies from specific human exposures. Discussion: Disease outcome is likely dependent on cell-type-specific responses and interaction with individual genetics, other toxicants, and infectious agents. Potential phenotypic anchors include target tissue dosimetry, gene expression and epigenetic profiles, and tissue biomarkers. Conclusions: Translation to human populations requires more extensive profiling of human samples along with high-quality dosimetry. Anchoring results by gene expression and epigenetic profiling has great promise for data unification. Genetic predisposition of individuals affects disease outcome. Interactions with infectious agents, particularly viruses, may explain some species-specific differences between human pathologies and experimental animal pathologies. Invertebrate systems amenable to genetic manipulation offer potential for elaborating impacts of specific biochemical pathways. Anchoring experimental results to specific human exposures will accelerate understanding of mechanisms of arsenic-induced human disease.
Arsenic is a lung toxicant that can lead to respiratory illness through inhalation and ingestion, although the most common exposure is through contaminated drinking water. Lung effects reported from arsenic exposure include lung cancer and obstructive lung disease, as well as reductions in lung function and immune response. As part of their role in innate immune function, airway epithelial cells provide a barrier that protects underlying tissue from inhaled particulates, pathogens, and toxicants frequently found in inspired air. We evaluated the effects of a five-day exposure to environmentally relevant levels of arsenic {4μM [~300 μg/L (ppb)] as NaAsO2} on airway epithelial barrier function and structure. In a primary mouse tracheal epithelial (MTE) cell model we found that both micromolar (3.9 μM) and submicromolar (0.8 μM) arsenic concentrations reduced transepithelial resistance, a measure of barrier function. Immunofluorescent staining of arsenic-treated MTE cells showed altered patterns of localization of the transmembrane tight junction proteins claudin (Cl) Cl-1, Cl-4, Cl-7 and occludin at cell-cell contacts when compared with untreated controls. To better quantify arsenic-induced changes in tight junction transmembrane proteins we conducted arsenic exposure experiments with an immortalized human bronchial epithelial cell line (16HBE14o-). We found that arsenic exposure significantly increased the protein expression of Cl-4 and occludin as well as the mRNA levels of Cl-4 and Cl-7 in these cells. Additionally, arsenic exposure resulted in altered phosphorylation of occludin. In summary, exposure to environmentally relevant levels of arsenic can alter both the function and structure of airway epithelial barrier constituents. These changes likely contribute to the observed arsenic-induced loss in basic innate immune defense and increased infection in the airway.
In this study, we examined the role of neprilysin (NEP), a key membrane-bound endopeptidase, in the inflammatory response induced by diesel exhaust emissions (DEE) in the airways through a number of approaches: in vitro, animal, and controlled human exposure. Our specific aims were (1) to examine the role of NEP in inflammatory injury induced by diesel exhaust particles (DEP) using Nep-intact (wild-type) and Nep-null mice; (2) to examine which components of DEP are associated with NEP downregulation in vitro; (3) to determine the molecular impact of DEP exposure and decreased NEP expression on airway epithelial cells' gene expression in vitro, using a combination of RNA interference (RNAi) and microarray approaches; and (4) to evaluate the effects on NEP activity of human exposure to DEE. We report four main results: First, we found that exposure of normal mice to DEP consisting of standard reference material (SRM) 2975 via intratracheal installation can downregulate NEP expression in a concentration-dependent manner. The changes were accompanied by increases in the number of macrophages and epithelial cells, as well as proinflammatory cytokines, examined in bronchoalveolar lavage (BAL) fluid and cells. Nep-null mice displayed increased and/or additional inflammatory responses when compared with wild-type mice, especially in response to exposure to the higher dose of DEP that we used. These in vivo findings suggest that loss of NEP in mice could cause increased susceptibility to injury or exacerbate inflammatory responses after DEP exposure via release of specific cytokines from the lungs. Second, we found evidence, using in vitro studies, that downregulation of NEP by DEP in cultured human epithelial BEAS-2B cells was mostly attributable to DEP-adsorbed organic compounds, whereas the carbonaceous core and transition metal components of DEP had little or no effect on NEP messenger RNA (mRNA) expression. This NEP downregulation was not a specific response to DEP or its contents because the change also occurred after exposure to urban dust (SRM 1649a), which differs in physical and chemical composition from DEP. Third, we also collected the transcriptome profiles of the concentration-effects of SRM 2975 in cultured BEAS-2B cells through a 2 X 3 factorial design. DEP exposure upregulated 151 genes and downregulated 59 genes. Cells with decreased NEP expression (accomplished by transfecting an NEP-specific small interfering RNA [siRNA]) substantially altered the expression of genes (upregulating 17 and downregulating 14) associated with DNA/protein binding, calcium channel activities, and the cascade of intracellular signaling by cytokines. Data generated from the combined RNAi and microarray approaches revealed that there is a complex molecular cascade mediated by NEP in different subcellular compartments, possibly influencing the inflammatory response. Fourth, in a controlled human exposure study, we observed significant increases in soluble NEP in sputum after acute exposure to DEE, with an average net increase of 31%. We speculate that the change in NEP activity in sputum, if confirmed in larger epidemiologic investigations at ambient exposure levels to DEE, may provide a useful endpoint and promote insight into the mechanism of DEE-induced airway alterations.