An In Vitro Versus In Vivo Toxicogenomic Investigation of Prenatal Exposures to Tobacco Smoke.

Approximately 1 million women smoke during pregnancy despite evidence demonstrating serious juvenile and/or adult diseases being linked to early-life exposure to cigarette smoke. Susceptibility could be determined by factors in previous generations, that is, prenatal or "maternal" exposures to toxins. Prenatal exposure to airborne pollutants such as mainstream cigarette smoke has been shown to induce early-life insults (i.e., gene changes) in Offspring that serve as biomarkers for disease later in life. In this investigation, we have evaluated genome-wide changes in the lungs of mouse Dams and their juvenile Offspring exposed prenatally to mainstream cigarette smoke. An additional lung model was tested alongside the murine model, as a means to find an alternative in vitro, human tissue-based replacement for the use of animals in medical research. Our toxicogenomic and bio-informatic results indicated that in utero exposure altered the genetic patterns of the fetus, which could put them at greater risk for developing a range of chronic illnesses in later life. The genes altered in the in vitro, cell culture model were reflected in the murine model of prenatal exposure to mainstream cigarette smoke. The use of alternative in vitro models derived from human medical waste tissues could be viable options to achieve human endpoint data and conduct research that meets the remits for scientists to undertake the 3Rs practices.

Gene and protein responses of human lung tissue explants exposed to ambient particulate matter of different sizes.

CONTEXT: Exposure to ambient particulate air pollution is associated with increased cardiovascular and respiratory morbidity and mortality. It is necessary to understand causal pathways driving the observed health effects, particularly if they are differentially associated with particle size. OBJECTIVES: To investigate the effect of different size ranges of ambient particulate matter (PM) on gene and protein expression in an in vitro model. MATERIALS AND METHODS: Normal human tracheobronchial epithelium (NHTBE) three-dimensional cell constructs were exposed for 24 h to washed ambient PM of different sizes (size 1: 7-615 nm; size 2: 616 nm-2.39 µm; size 3: 2.4-10 µm) collected from a residential street. A human stress and toxicity PCR array was used to investigate gene expression and iTRAQ was used to perform quantitative proteomics. RESULTS: Eighteen different genes of the 84 on the PCR array were significantly dysregulated. Treatment with size 2 PM resulted in the greatest number of genes with altered expression, followed by size 1 and lastly size 3. ITRAQ identified 317 proteins, revealing 20 that were differentially expressed. Enrichment for gene ontology classification revealed potential changes to various pathways. DISCUSSION AND CONCLUSIONS: Different size fractions of ambient PM are associated with dysregulatory effects on the cellular proteome and on stress and toxicity genes of NHTBE cells. This approach not only provides an investigative tool to identify possible causal pathways but also permits the relationship between particle size and responses to be explored.

Proteomic profiling of human respiratory epithelia by iTRAQ reveals biomarkers of exposure and harm by tobacco smoke components.

Historically, it has been challenging to go beyond epidemiology to investigate the pathogenic changes caused by tobacco smoking. The EpiAirway-100 (MatTek Corp., Ashland, MA) was employed to investigate the effects of cigarette smoke components. Exposure at the air-liquid-interface represented particle and vapour phase components of cigarette smoke. A proteomic study utilising iTRAQ labelling compared expression profiles. The correlative histopathology revealed focal regions of hyperplasia, hypertrophy, cytolysis and necrosis. We identified 466 proteins, 250 with a parameter of two or more peptides. Four of these proteins are potential markers of lung injury and three are related to mechanistic pathways of disease.

The TNF-like protein 1A-death receptor 3 pathway promotes macrophage foam cell formation in vitro.

TNF-like protein 1A (TL1A), a TNF superfamily cytokine that binds to death receptor 3 (DR3), is highly expressed in macrophage foam cell-rich regions of atherosclerotic plaques, although its role in foam cell formation has yet to be elucidated. We investigated whether TL1A can directly stimulate macrophage foam cell formation in both THP-1 and primary human monocyte-derived macrophages with the underlying mechanisms involved. We demonstrated that TL1A promotes foam cell formation in human macrophages in vitro by increasing both acetylated and oxidized low-density lipoprotein uptake, by enhancing intracellular total and esterified cholesterol levels and reducing cholesterol efflux. This imbalance in cholesterol homeostasis is orchestrated by TL1A-mediated changes in the mRNA and protein expression of several genes implicated in the uptake and efflux of cholesterol, such as scavenger receptor A and ATP-binding cassette transporter A1. Furthermore, through the use of virally delivered DR3 short-hairpin RNA and bone marrow-derived macrophages from DR3 knockout mice, we demonstrate that DR3 can regulate foam cell formation and contributes significantly to the action of TL1A in this process in vitro. We show, for the first time, a novel proatherogenic role for both TL1A and DR3 that implicates this pathway as a target for the therapeutic intervention of atherosclerosis.

Airborne particles in Swansea, UK: their collection and characterization.

Urban air particulate matter (PM) has previously been associated with a variety of adverse health effects. It is now believed that the smallest particles, ultrafine or nanoparticles, are linked to the greatest health effects. The physicochemistry of these particles is likely to provide information regarding their toxicity. Therefore, the aim of this study was to further the understanding of the heterogeneous and changing particle concentrations in urban air, in conjunction with gaining an understanding of the physicochemistry of the particles. A Dekati electrical low-pressure impactor was used to collect the particles and real-time data in a busy traffic corridor in Swansea, Wales, over a period of 10 nonconsecutive weeks. Particle concentrations in the street canyon were analyzed and particle physicochemistries investigated using a variety of techniques. Particle number concentrations were found to vary both diurnally and from day to day in the traffic corridor. Of all particles, the nano to fine size fraction was consistently identified in the highest concentrations (maximum: 140,000 particles cm(-3)). Particle physicochemistry was found to vary as a function of size, with larger particles exhibiting a greater variety of morphologies (and consequently particle types) and associated metals.

A novel application for Cocoacrisp protein as a biomarker for experimental pulmonary fibrosis.

Pulmonary fibrosis is a debilitating disease affecting up to 2 million people worldwide, with a median survival rate of only 3 years after diagnosis. The aim of this study was to evaluate a potential protein biomarker (Cocoacrisp, CC) to identify the onset of pulmonary fibrosis. A model of fibrosis was induced via intratracheal instillation of bleomycin, and samples were collected during the early phase of the disease. Immunohistochemical identification of CC was carried out in lung tissue from the bleomycin model. Quantification by image analysis showed CC levels were doubled (p <0.0003), after a single bleomycin dose, but not after double instillation. Microscopic analysis revealed that CC signal was primarily detected on the alveolar surface. The secretion of the novel protein CC during the early stages of bleomycin-induced injury may have the potential to be utilized as a clinical biomarker for the early stages of fibrosis, particularly as it may be detectable in bronchoalveolar lavage fluid.

Genomic biomarkers of pulmonary exposure to tobacco smoke components.

BACKGROUND: Associations between smoking and the development of tobacco-related diseases in humans have historically been assessed by epidemiological studies. These studies are further complicated by the number of chemicals used in tobacco and individual smoking habits. An alternative approach is required to assess the biological responses. OBJECTIVE: Toxicogenomics was carried out to identify early molecular markers for events in pulmonary injury resulting from tobacco smoke components (TSC) exposure. MATERIALS AND METHODS: EpiAirway-100 cells were exposed at the air/liquid interface to representative particle (nicotine; cadmium) and vapour phase [formaldehyde (FA) and ethyl carbamate] components of cigarette smoke. Microarray technology was used to compare expression profiles of human genes associated with toxicity and drug resistance, from control and TSC-treated respiratory epithelium (n=5/dose). RESULTS: Using the GEArray 'toxicology and drug resistance' microarray followed by significance analysis of microarray analysis, 42 mRNA transcripts were found to be significantly altered by the TSC exposure. The vapour [ethyl carbamate, FA and particle (nicotine, cadmium)] phase TSC exhibited differential transcriptional responses that could not be attributed to their chemical phase. The transcriptional changes could be classified according to a functional family, where ethyl carbamate, FA and cadmium classified as carcinogens, demonstrated the highest gene homology when compared with the noncarcinogen, nicotine. DISCUSSION: Analysis of the microarray data and further confirmation (reverse transcriptase-PCR) identified three potential biomarkers for TSC-induced injury. These three genes (CYP7A1, HMOX1 and PTGS1) are highly upregulated and have been linked with mechanistic pathways of disease.

An in vitro approach to assess the toxicity of inhaled tobacco smoke components: nicotine, cadmium, formaldehyde and urethane.

One of the first lines of defence to inhaled toxins is the barrier formed by the tracheobronchial epithelium, making this the ideal region for studying the toxicity of inhaled substances. This study utilises a highly differentiated, three-dimensional, in vitro model of human upper respiratory tract epithelium (EpiAirway-100) to measure the acute toxicological responses to well-characterised tobacco smoke components. To determine the suitability of this model for screening inhaled toxicants, the EpiAirway tissue model (ETM) was treated apically with tobacco smoke components (nicotine, formaldehyde, cadmium, urethane) which are known to induce a variety of toxic effects (e.g. cytotoxic, thrombogenic, carcinogenic). A range of concentrations were used to model different mechanisms and severity of toxicity which were then compared to known in vivo responses. Similar trends in stress response occurred, with distinct alterations to the tissue in response to all four toxins. At high concentrations, cell viability decreased and tight junctions were degraded, but at sub-toxic concentrations epithelial resistance (indicating tissue integrity) increased 20-60% from control. This peak in resistance coincided with an increase in secreted protein levels, elevated cytokine release and goblet cell hyperplasia and hypertrophy. In conclusion, acute exposure to tobacco smoke components induces measurable toxic responses within human respiratory epithelium. Sub-toxic concentrations appear to illicit a protective response by increasing mucus secretion and mediating immune responses via cytokine release. These responses are comparable to human in vivo responses, indicating potential for the ETM as a tool for screening the toxicity of inhaled compounds.

Combustion-derived nanoparticles: mechanisms of pulmonary toxicity.

1. The general term 'nanoparticle' (NP) is used to define any particle less than 100 nm in at least one dimension and NPs are generally classified as natural, anthropogenic or engineered in origin. Anthropogenic, also referred to as 'ultrafine' particles (UFPs), are predominately combustion derived and are characterized by having an equivalent spherical diameter less than 100 nm. 2. These particles, considered to be 'combustion-derived nanoparticles' (CDNPs), are of toxicological interest given their nanosized dimensions, with properties not displayed by their macroscopic counterparts. 3. The pulmonary deposition efficiency of inhaled UFPs, along with their large surface areas and bound transition metals, is considered important in driving the emerging health effects linked to respiratory toxicity. 4. The toxicology of CDNPs is currently used to predict the health outcomes in humans following exposure to manufactured NPs. Their similar physicochemistry would suggest similar adverse health effects (i.e. pulmonary (and perhaps cardiac) toxicity). As such, it is essential to fully understand CDNP nanotoxicology in order to minimize occupational and environmental exposure.

Characterisation of airborne particles collected within and proximal to an opencast coalmine: South Wales, U.K.

Airborne particulate matter has been collected from within, and proximal to, an opencast coal mine in south Wales. This work forms the first part of a three year project to collect and characterise, then determine the possible toxicology of airborne particles in the south Wales region. High-resolution Field Emission SEM has shown that the coal mine dusts consist largely of an assemblage of mineral grains and vehicle exhaust particles. SEM-EDX has shown that the mineralogical make-up of the PM10 is complex, heterogeneous, and constantly changing. These findings are supported by analytical TEM-EPXMA. However, patterns can be determined relating the mineralogical composition of the airborne particles to collection locations and mining activities within the opencast. At our study opencast, Park Slip West, quartz, which has known health effects, never exceeded 30% of the total collection mass, and average levels were much less. Vehicle exhaust emissions was the largest source in terms of particle numbers. The mass of airborne particulate matter within the pit averaged approximately twice that of outside the pit: importantly however, this higher mass was due to relatively large, and non-respirable, mineral grains. This study demonstrates that the physicochemical and mineralogical characterisation of airborne particles from mining and quarrying is essential to quantify the respirable fraction, and to identify potentially hazardous components within the PM10.