LEY 26505 PDF

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Quantitative real-time PCR analysis of a representative set of 10 genes confirmed the microarray findings. The number of biological functions, canonical pathways and molecular networks significantly affected by silica exposure, as identified by the bioinformatics analysis of the significantly differentially expressed genes detected during the post-exposure time intervals, also exhibited a steady increase similar to the silica-induced pulmonary toxicity.

Genes involved in oxidative stress, inflammation, respiratory diseases, cancer, and tissue remodeling and fibrosis were significantly differentially expressed in the rat lungs; however, unresolved inflammation was the single most significant biological response to pulmonary exposure to silica.

Excessive mucus production, as implicated by significant overexpression of the pendrin coding gene, SLC26A4oey identified as a potential novel mechanism for silica-induced pulmonary toxicity.

Collectively, the findings of our study provided insights into the molecular mechanisms underlying the progression of crystalline silica-induced pulmonary toxicity in the rat. Exposure to respirable crystalline silica silica takes place pey a variety of industries and occupational settings because of its extremely common natural occurrence and the wide range of materials and products that contain it. Virtually any process that involves the movement of earth or disturbance of products such as concrete and masonry may expose workers to silica.

It is estimated that at least 1. In lfy to silicosis, a life-threatening lung pneumoconiosis, occupational exposure to silica is associated with the development of bronchitis, emphysema, tuberculosis, systemic sclerosis, rheumatoid arthritis, lupus, chronic renal disease and lung cancer IARC, ; Cooper et al.

A proper understanding of the molecular targets and mechanisms underlying the initiation and progression of silica-induced pulmonary toxicity is required to develop strategies potentially to prevent the various diseases associated with silica exposure.

In spite of the large number of studies conducted in the past investigating the toxicity of crystalline silica, neither the molecular targets nor the mechanisms underlying its toxicity are fully understood. Advances in high-throughput gene expression profiling, such as microarray analysis, enable a comprehensive understanding of the effects lry toxic agents at the molecular level in biological systems.

The gene expression data, in addition, may be useful to generate novel hypotheses regarding the molecular mechanisms underlying the toxicity of the agent being investigated. In the past, microarray-based transcriptomics studies have been successfully employed to gain insights into the molecular mechanisms underlying the toxicity of chemicals Waring et al.

In addition, it has been fairly well established that gene expression changes relevant to toxicity precede biochemical and histological changes indicative of target organ toxicity Foster et al.

Recently, key have reported developing a rat model for silica-induced pulmonary toxicity Sellamuthu et al. We determined the global gene expression profile in the lungs obtained from these rats to identify the molecular targets as well as to understand the mechanisms involved in silica-induced pulmonary toxicity progression.

Bioinformatics analysis of the gene expression data identified molecular targets of silica toxicity and provided insights into the molecular ldy underlying the lwy of silica-induced pulmonary toxicity in the rats. Throughout the period of the experiment the rats were maintained on a 12 h light—dark schedule with free access to rat diet Harlan Laboratories, Frederick, MD, USA and tap water.

A thorough characterization of the silica particles generated and employed in this inhalation exposure study was performed. Representative samples collected from the inhalation exposure chamber were analyzed for particle morphology by scanning electron microscopy. Results of these analyses can be found in our recent publication Sellamuthu et al. Commercial sources of all other reagents used in this study are provided in the corresponding sections below.

Details regarding generation of the crystalline silica aerosol and inhalation exposure of rats to the aerosol have been published previously Sellamuthu et al. A Venturi was placed in the inhalation exposure system in between the acoustic generator and the exposure chamber to prevent the agglomeration of silica particles generated. Forty rats exposed simultaneously to filtered air served as the controls.

The lung samples from the control and silica-exposed rats were collected to determine pulmonary toxicity and the findings have been reported recently Sellamuthu et al.

Simultaneously, RNA was isolated from the lung samples to determine global gene expression profile as described below. After centrifugation at 10 g for 3 min at room temperature, the pey containing RNA was isolated and applied 2605 the RNeasy column and processed as directed in the RNeasy Fibrous Tissue Mini Kit protocol. Chip hybridizations, washing, Cy3-streptavidin staining and scanning of the chips on the Beadstation platform Illumina Inc.

Metric files from the bead scanner were checked to ensure that all samples fluoresced at comparable levels before samples were loaded into Beadstudio Framework version 3.

Housekeeping, hybridization control, stringency and negative control genes were checked for proper chip detection. BeadArray expression data were then exported with mean fluorescent intensity across like beads and bead variance estimates into flat files for subsequent analysis.

Bioconductor is a project for the analysis and comprehension of genomic data and operates in R, a statistical computing environment Ihaka and Gentleman, In short, limma fits a linear model for each gene, generates group means of expression and calculates P -values and log fold-changes which are converted to standard fold changes.

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The specificity and integrity of the PCR products were determined by analyzing the melting curves of all PCR amplified gene products.

IPA software is designed to map the biological relationship of the uploaded genes and classify them into categories of biological functions, molecular networks or canonical pathways according to published literature in the database.

Nonmicroarray data between the silica-exposed and corresponding time-matched control group of rats were compared using the one-way ANOVA test.

A summary of 226505 recently published findings on silica induced pulmonary toxicity in the rats employed in this study is presented in Table 2. The silica-induced pulmonary toxicity, in general, exhibited a steady progression during the post-exposure time intervals analyzed as evidenced from the various biochemical, histological and cellular toxicity parameters determined in the rats.

Molecular insights into the progression of crystalline silica-induced pulmonary toxicity in rats

Summary of the pulmonary toxicity evaluation findings of crystalline silica exposed rats adapted from Sellamuthu et al. Microarray analysis of the global gene expression profile identified the genes whose ,ey were significantly affected by silica exposure in the lungs of rats Supporting Information, tables 1 — 5. The number of SDEGs identified in le lungs of the silica-exposed rats, compared with the corresponding time-matched control rats, exhibited a steady increase during the post-exposure time intervals analyzed Fig.

The increase in the number lej SDEGs in leey silica-exposed rats exhibited a trend similar to the various pulmonary toxicity parameters observed during the post-exposure time intervals. The number 62505 genes differentially leu totaloverexpressed up and under expressed down in the silica exposed rat lungs compared with the corresponding time-matched controls are presented for the post-exposure lej intervals presented on the X-axis. Data represents the mean of eight silica exposed rats compared with four corresponding time-matched control rats per time point.

In general, the progressive increase in the magnitude of overexpression of most of the representative genes observed in the silica-exposed ldy lungs during the post-exposure time intervals was confirmed by the results of QRT-PCR analysis. A set of 10 genes which were significantly differentially expressed in the silica exposed rat lungs as evidenced from the microarray data presented in Figure 2A was analyzed by QRT-PCR as described in the Materials and methods section and the results are presented in Figure 2B.

Bioinformatics analysis of the SDEGs obtained from the microarray analysis identified the various biological functions, canonical pathways and molecular networks that were significantly enriched in the rat lungs by inhalation exposure to silica.

The top ranking biological functions significantly affected by silica exposure were inflammatory response, cell-to-cell signaling and interaction, cellular movement, inflammatory diseases, respiratory diseases and cancer Fig. The number of SDEGs belonging to each of these top ranking biological functions, as in the case of silica-induced pulmonary toxicity Table 2also exhibited a steady increase during the post-exposure time intervals analyzed Fig.

The vast majority of the significantly enriched canonical pathways in the silica-exposed rat lungs were those involved in an inflammatory response Supporting Information, table 6. The time-course of enrichment of acute phase response and complement system in the silica-exposed rat lungs during the post-exposure time intervals are presented as representative canonical pathways enriched by silica exposure in the rats Fig.

The involvement of both these canonical 2605, as evidenced by their IPA P ldy, in the pulmonary response of the silica-exposed rat lungs also exhibited a steady increase during the post-exposure time intervals analyzed. The number of molecular networks significantly enriched in the rat lungs in response to pulmonary exposure to silica Fig. A selected list of SDEGs belonging to the various biological functions, pathways and networks that are significantly enriched and, therefore, are considered to be of importance in the silica-induced pulmonary toxicity is presented in Table 3 and the functional significance of their differential expression with respect to the progression of silica-induced pulmonary toxicity is discussed below.

Bioinformatics analysis of the significantly differentially expressed genes in the silica exposed rat lungs was done using IPA software. Data represents the group mean of eight silica exposed and four time-matched control rats per time point.

Lye number of significantly differentially expressed genes SDEGs in the silica exposed rat lungs belonging to the six top ranking IPA biological functions at each of the post-exposure time interval is presented. The complement system A and acute phase response key B are presented as representative IPA canonical pathways enriched in the silica exposed rat lungs.

Proyecto de Ley 2462/2012-CR

A complete listing of the IPA canonical pathways enriched in rat lungs in response to silica exposure is presented in Supporting information, table 6.

The numbers presented in the parenthesis adjacent to the gene symbols are the silica post-exposure time interval in weeks at which the gene represented was significantly differentially expressed in the silica exposed rat lungs compared with the corresponding time-matched control rats.

NR1D1 expression was significantly reduced while all other genes let significantly overexpressed in the silica exposed rat lungs. Fold change in expression of a selected list of significantly differentially expressed genes in the lungs of silica 25605 rats.

Some of the genes are listed under more than one category ely bioinformatics analysis identified their involvement in multiple categories. A comprehensive understanding of the molecular mechanisms underlying the initiation and leu of silica-induced pulmonary toxicity, which is critical in the potential prevention of diseases associated with silica exposure, is still lacking.

The present study is part of an on-going research project aiming to identify the molecular targets and mechanisms lwy silica-induced pulmonary toxicity. The silica-induced pulmonary toxicity in these rats, in agreement with previous reports Johnston et al.

Presently, the lung samples obtained from these rats were analyzed by microarray to determine a global gene expression profile in order to identify the molecular targets as well as to elucidate the molecular mechanisms underlying the progression of silica-induced pulmonary toxicity.

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The rat model for silica-induced pulmonary toxicity employed in this study is relevant to human silicosis. Analogous to the human situation, progression of silica-induced pulmonary toxicity for a prolonged period after cessation of silica exposure was observed in the rat model employed in this study Table 2. The results presented in this report justified the application of global gene expression profiling as a relevant approach to identify the molecular targets as well as to elucidate the molecular mechanisms underlying the progression of silica-induced pulmonary toxicity.

As presented in Table 4the various pulmonary toxicity parameters correlated well with the gene expression findings in the silica-exposed rats. In addition, results of the bioinformatics analysis of the SDEGs, in agreement with the findings of ly previous studies, reaffirmed the ability of silica exposure to result in the induction of inflammation Barbarin et al. Correlation co-efficients r 2 values for the relationship between 2505 toxicity and inflammation measurements LDH, PMN and MCP1 and lung gene expression data in the silica exposed rats.

Generation of reactive oxygen species directly from silica particles Vallyathan et al. This argument is further supported by the significant overexpression of LPOan H 2 O 2 -responsive gene Davies et al.

A central role for inflammation in the pulmonary effects associated with silica exposure has been established Castranova, Significant increase in the number of AMs and PMNs and concentrations of the pro-inflammatory chemokines, MCP1 and MIP2, noticed in the lung samples used in this study Table 2suggested the lry of significant pulmonary inflammation in our rat model.

The silica-induced pulmonary inflammation in the pey, similar to the trend exhibited by the various parameters of silica-induced pulmonary toxicity, exhibited a steady progression during the post-exposure time intervals analyzed Table 2. Bioinformatics analysis of the SDEGs supported the induction and progression of pulmonary inflammation and toxicity noticed in the silica-exposed rats.

Inflammatory response, inflammatory diseases and cellular movement were three of the top ranking IPA biological functions identified as being significantly enriched by silica exposure in the rat lungs Fig. Interestingly, the number of inflammation-related biological functions, pathways and networks that were significantly affected by silica exposure in the lungs also steadily increased Figs 4 — 6 ely with the progression of silica-induced pulmonary toxicity in the rats Table 2suggesting a possible relationship between silica-induced differential expression of genes involved in inflammation and the toxicity progression noticed in the rat lungs.

Gene expression profiling and bioinformatics analysis of the SDEGs also provided insights into the molecular mechanisms underlying the progression of silica-induced pulmonary inflammation and toxicity in the rats. It is noteworthy that overexpression of all these inflammatory response genes steadily increased along with the progression of silica-induced pulmonary inflammation and toxicity in the rats during the post-exposure time intervals analyzed, further supporting their involvement in the progression of pulmonary inflammation and toxicity in the silica-exposed rats.

Lipoxins play an important role in the resolution of pulmonary inflammation Chan and Moore,and the involvement of lipoxins, if any, in silica-induced pulmonary inflammation has not been investigated to date. Even though the pulmonary level of lipoxins was not measured in the silica-exposed rats, our gene expression data provided indirect evidence for the involvement of lipoxins in silica-induced pulmonary inflammation.

Lipoxins are products of arachidonic acid metabolism catalyzed by lipoxygenase Alox15; Kronke et al. An anti-inflammatory role has been attributed to lipoxins mainly because of their ability to inhibit chemotaxis, adhere and transmigrate neutrophils and antagonize the pro-inflammatory effects of leukotriens Colgan et al. Alox expression was significantly lower in the lungs of the silica-exposed rats compared with the time-matched controls Table 3.

Therefore, it is reasonable to assume that, in addition to the significant overexpression of the multiple pro-inflammatory genes, the significant down-regulation of Alox gene expression might have contributed to the establishment of unresolved pulmonary inflammation noticed in the silica-exposed rats.

Pulmonary fibrosis is a major component of silicosis Ng and Chan,the most serious health outcome of occupational exposure to silica. Even leey significant histological pre-fibrotic changes, including type II pneumocyte hyperplasia, occurred in the rat lungs at 16 weeks following cessation of silica exposure Table 2pulmonary fibrosis had not developed at this stage.

However, after a prolonged post-exposure time interval of 32 weeks, positive trichrome staining indicative of pulmonary fibrosis was observed in the 25605 rat lungs unpublished data. Bioinformatics analysis of the gene expression data, on the other hand, identified significant differential expression of several genes involved in tissue remodeling and fibrosis in the lungs of the silica-exposed rats as early as one week after cessation of silica exposure Table 3.

Matrix metalloproteinases MMPs are a family of proteins participating in many normal biological processes as well as in pathological processes, including fibrotic lung diseases Nagase and Woessner, A definite role for MMP12 in the induction of pulmonary fibrosis has been demonstrated previously in mice carrying a targeted deletion of the MMP12 gene Matute-Bello et al. Osteopontin, one of the key components of extracellular matrix, 266505 the migration, adhesion and proliferation of fibroblasts culminating in pulmonary fibrosis Takahashi et al.

The profibrotic gene Leyywhich codes osteopontin protein, was significantly overexpressed in the silica-exposed rat lungs with increased overexpression at late post-exposure time intervals of 8 and 16 weeks Table 3.