Influence of sex and disease severity on gene expression profiles in individuals with idiopathic pulmonary fibrosis.

Epidemiological studies suggest sex-specific trends in the prevalence and mortality of idiopathic pulmonary fibrosis (IPF) and chronic obstructive pulmonary disease (COPD) that are distinct for each disease. While the expression of numerous immune and extracellular matrix (ECM) genes in the lung have been well characterized in these diseases, associations elucidating their sex-specific expression patterns by disease type and severity, and the evaluation of hormone-related genes, have not been well studied. Here we performed targeted transcriptional profiling of 48 genes was performed on lung tissue samples from males and females with mild or medium severity IPF or COPD. The genes assessed included those involved in inflammation, ECM remodeling and hormonal processes. Data for 36 lung tissue samples were obtained that were stratified by disease and sex. Expression levels revealed a subset of genes which show differential expression among sexes, disease type, and disease severity. The most significant observations were the increased expression primarily of ECM genes in medium severity IPF (CATHK, COL1A1, COL3, MMP1, MMP7, IL-1RN) compared to mild IPF and COPD. Two genes, CH3L1 and MMP7 showed a tendency of interaction between sex and disease in IPF severity. Surprisingly, there were no significant differences in any of the sex genes measured between the IPF groups; however, ESR1 and AR expression levels were higher and lower, respectively, compared to COPD samples. Overall, this work highlights two genes, CH3L1 and MMP7, that may contribute to gender trends observed for IPF and COPD and are potential targets for future research.

Aryl phosphate esters within a major PentaBDE replacement product induce cardiotoxicity in developing zebrafish embryos: potential role of the aryl hydrocarbon receptor.

Firemaster 550 (FM550) is an additive flame retardant formulation of brominated and aryl phosphate ester (APE) components introduced as a major replacement product for the commercial polybrominated diphenyl ether mixture (known as PentaBDE) used primarily in polyurethane foam. However, little is known about the potential effects of FM550-based ingredients during early vertebrate development. Therefore, we first screened the developmental toxicity of each FM550 component using zebrafish as an animal model. Based on these initial screening assays, we found that exposure to the brominated components as high as 10µM resulted in no significant effects on embryonic survival or development, whereas exposure to triphenyl phosphate (TPP) or mono-substituted isopropylated triaryl phosphate (mono-ITP)-two APEs comprising almost 50% of FM550-resulted in targeted effects on cardiac looping and function during embryogenesis. As these cardiac abnormalities resembled aryl hydrocarbon receptor (AHR) agonist-induced phenotypes, we then exposed developing embryos to TPP or mono-ITP in the presence or absence of an AHR antagonist (CH223191) or AHR2-specific morpholino. Based on these studies, we found that CH223191 blocked heart malformations following exposure to mono-ITP but not TPP, whereas AHR2 knockdown failed to block the cardiotoxic effects of both components. Finally, using a cell-based human AHR reporter assay, we found that mono-ITP (but not TPP) exposure resulted in a significant increase in human AHR-driven luciferase activity at similar nominal concentrations as a potent reference AHR agonist (ß-naphthoflavone). Overall, our findings suggest that two major APE components of FM550 induce severe cardiac abnormalities during early vertebrate development.

Adverse outcome pathways during zebrafish embryogenesis: a case study with paraoxon.

Using paraoxon as a reference acetylcholinesterase (AChE) inhibitor, the objective of this study was to develop an adverse outcome pathway (AOP) that provided quantitative linkages across levels of biological organization during zebrafish embryogenesis. Within normal zebrafish embryos, we first demonstrated that ache transcripts and AChE activity increased in a stage-dependent manner following segmentation. We then showed that static exposure of embryos to paraoxon (31.2-500 nM) from 5 to 96 hpf resulted in significant stage- and concentration-dependent AChE inhibition, albeit these effects were fully reversible within 48 h following transfer to clean water. However, even in the presence of significant AChE inhibition, exposure to non-teratogenic paraoxon concentrations (≤250 nM) did not adversely impact secondary motoneuron development at 96 hpf. Therefore, we investigated the potential effects of paraoxon exposure on spontaneous tail contractions at 26 hpf - an early locomotor behavior that results from innervation of primary (not secondary) motoneuron axons to target axial muscles. Based on these studies, the frequency of spontaneous tail contractions at 26 hpf - a developmental stage with minimal AChE expression and activity - was significantly higher following exposure to paraoxon concentrations as low as 31.2 nM. Overall, our data suggest that (1) normal AChE activity is not required for secondary motoneuron development and (2) spontaneous tail contractions at 26 hpf are sensitive to paraoxon exposure, an effect that may be independent of AChE inhibition. Using a well-studied reference chemical, this study highlights the potential challenges in developing quantitative AOPs to support chemical screening and prioritization strategies.

Early zebrafish embryogenesis is susceptible to developmental TDCPP exposure.

BACKGROUND: Chlorinated phosphate esters (CPEs) are widely used as additive flame retardants for low-density polyurethane foams and have frequently been detected at elevated concentrations within indoor environmental media. OBJECTIVES: To begin characterizing the potential toxicity of CPEs on early vertebrate development, we examined the developmental toxicity of four CPEs used in polyurethane foam: tris(1,3-dichloro-2-propyl) phosphate (TDCPP), tris(2-chloroethyl) phosphate (TCEP), tris(1-chloro-2-propyl) phosphate (TCPP), and 2,2-bis(chloromethyl)propane-1,3-diyl tetrakis(2-chlorethyl) bis(phosphate) (V6). METHODS: Using zebrafish as a model for vertebrate embryogenesis, we first screened the potential teratogenic effects of TDCPP, TCEP, TCPP, and V6 using a developmental toxicity assay. Based on these results, we focused on identification of susceptible windows of developmental TDCPP exposure as well as evaluation of uptake and elimination of TDCPP and bis(1,3-dichloro-2-propyl)phosphate (BDCPP, the primary metabolite) within whole embryos. Finally, because TDCPP-specific genotoxicity assays have, for the most part, been negative in vivo and because zygotic genome remethylation is a key biological event during cleavage, we investigated whether TDCPP altered the status of zygotic genome methylation during early zebrafish embryogenesis. RESULTS: Overall, our findings suggest that the cleavage period during zebrafish embryogenesis is susceptible to TDCPP-induced delays in remethylation of the zygotic genome, a mechanism that may be associated with enhanced developmental toxicity following initiation of TDCPP exposure at the start of cleavage. CONCLUSIONS: Our results suggest that further research is needed to better understand the effects of a widely used and detected CPE within susceptible windows of early vertebrate development.

Osteopontin modulates inflammation, mucin production, and gene expression signatures after inhalation of asbestos in a murine model of fibrosis.

Inflammation and lung remodeling are hallmarks of asbestos-induced fibrosis, but the molecular mechanisms that control these events are unclear. Using laser capture microdissection (LCM) of distal bronchioles in a murine asbestos inhalation model, we show that osteopontin (OPN) is up-regulated by bronchiolar epithelial cells after chrysotile asbestos exposures. In contrast to OPN wild-type mice (OPN(+/+)) inhaling asbestos, OPN null mice (OPN(-/-)) exposed to asbestos showed less eosinophilia in bronchoalveolar lavage fluids, diminished lung inflammation, and decreased mucin production. Bronchoalveolar lavage fluid concentrations of inflammatory cytokines (IL-1ß, IL-4, IL-6, IL-12 subunit p40, MIP1α, MIP1ß, and eotaxin) also were significantly less in asbestos-exposed OPN(-/-) mice. Microarrays performed on lung tissues from asbestos-exposed OPN(+/+) and OPN(-/-) mice showed that OPN modulated the expression of a number of genes (Col1a2, Timp1, Tnc, Eln, and Col3a1) linked to fibrosis via initiation and cross talk between IL-1ß and epidermal growth factor receptor-related signaling pathways. Novel targets of OPN identified include genes involved in cell signaling, immune system/defense, extracellular matrix remodeling, and cell cycle regulation. Although it is unclear whether the present findings are specific to chrysotile asbestos or would be observed after inhalation of other fibers in general, these results highlight new potential mechanisms and therapeutic targets for asbestosis and other diseases (asthma, smoking-related interstitial lung diseases) linked to OPN overexpression.

Gender influences the response to experimental silica-induced lung fibrosis in mice.

Accumulating evidence suggests that gender can have a profound effect on incidence and severity of a variety of pulmonary diseases. To address the influence of gender on the development of silica-induced pulmonary fibrosis, we instilled 0.2 g/kg silica into male and female C57BL/6 mice and examined the fibrotic and inflammatory response at 14 days postexposure. Both silica-exposed male and female mice had significant increases in total lung hydroxyproline compared with saline controls. However, silica-exposed female mice had significantly less total lung hydroxyproline than silica-exposed male mice. This observation was confirmed by color thresholding image analysis. Interestingly, silica-exposed female mice had significantly more inflammatory cells, the majority of which were macrophages, as well as higher levels of the macrophage-specific chemokines MCP-1 and CCL9 in whole lung lavage compared with silica-exposed male mice. We also show that at baseline, estrogen receptor α (ERα) mRNA expression is lower in female mice than in males and that ERα mRNA expression is decreased by silica exposure. Finally, we show that the response of ovariectomized female mice to silica instillation is similar to that of male mice. These observations together show that gender influences the lung response to silica.