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Nanomaterial science and design have shifted from generating single passive nanoparticles to more complex and adaptive multi-component nanohybrids. These adaptive nanohybrids (ANHs) are designed to simultaneously perform multiple functions, while actively responding to the surrounding environment. ANHs are engineered for use as drug delivery carriers, in tissue-engineered templates and scaffolds, adaptive clothing, smart surface coatings, electrical switches and in platforms for diversified functional applications. Such ANHs are composed of carbonaceous, metallic or polymeric materials with stimuli-responsive soft-layer coatings that enable them to perform such switchable functions. Since ANHs are engineered to dynamically transform under different exposure environments, evaluating their environmental behavior will likely require new approaches. Literature on polymer science has established a knowledge core on stimuli-responsive materials. However, translation of such knowledge to environmental health and safety (EHS) of these ANHs has not yet been realized. It is critical to investigate and categorize the potential hazards of ANHs, because exposure in an unintended or shifting environment could present uncertainty in EHS. This article presents a perspective on EHS evaluation of ANHs, proposes a principle to facilitate their identification for environmental evaluation, outlines a stimuli-based classification for ANHs and discusses emerging properties and dynamic aspects for systematic EHS evaluation.

Previous studies indicate that exposure of fish to pristine single-walled carbon nanotubes (SWCNTs) by oral gavage, causes no overt toxicity, and no appreciable absorption has been observed. However, in the environment, SWCNTs are likely to be present in dietary sources, which may result in differential impacts on uptake and biological effects. Additionally, the potential of these materials to sorb nutrients (proteins, carbohydrates, and lipids) while present in the gastrointestinal (GI) tract may lead to nutrient depletion conditions that impact processes such as growth and reproduction. To test this phenomenon, fathead minnows were fed a commercial diet either with or without SWCNTs for 96 h. Tracking and quantification of SWCNTs using near-infrared fluorescence (NIRF) imaging during feeding studies showed the presence of food does not facilitate transport of SWCNTs across the intestinal epithelia. Targeting genes shown to be responsive to nutrient depletion (peptide transporters, peptide hormones, and lipases) indicated that pept2 , a peptide transporter, and cck , a peptide hormone, showed differential mRNA expression by 96 h, a response that may be indicative of nutrient limitation. The results of the current study increase our understanding of the movement of SWCNTs through the GI tract, while the changes in nutrient processing genes highlight a novel mechanism of sublethal toxicity in aquatic organisms.

Single-walled carbon nanotubes' (SWNT) effectiveness in applications is enhanced by debundling or stabilization. Anionic surfactants are known to effectively stabilize SWNTs. However, the role of specific chirality on surfactant-stabilized SWNT aggregation has not been studied to date. The aggregation behavior of chirally enriched (6,5) and (7,6) semiconducting SWNTs, functionalized with three anionic surfactants-sodium dodecyl sulfate (SDS), sodium dodecyl benzene sulfonate (SDBS), and sodium deoxycholate (SDOCO)-was evaluated with time-resolved dynamic light scattering. A wide range of mono- (NaCl) and di-valent (CaCl 2 ) electrolytes as well as a 2.5 mg TOC/L Suwannee River humic acid (SRHA) were used as background chemistry. Overall, SDBS showed the most effectiveness in SWNT stability, followed by SDOCO and SDS. However, the relatively larger diameter (7,6) chiral tubes compromised the surfactant stability, compared to (6,5) chiral enrichment, due to enhanced van der Waals interaction. The presence of di-valent electrolytes overshadowed the chirality effects and resulted in similar aggregation behavior for both the SWNT samples. Molecular modeling results enumerated key differences in surfactant conformation on SWNT surfaces and identified interaction energy changes between the two chiralities to delineate aggregation mechanisms. The stability of SWNTs increased in the presence of SRHA under 10 mM monovalent and mixed electrolyte conditions. The results suggest that change in chirality can overcome surfactant stabilization of semiconducting SWNTs. SWNT stability can also be strongly influenced by the anionic surfactant structure.

Background: Airborne exposure to nanomaterials from unintended occupational or environmental exposures or as a consequence of product use may lead to adverse health effects. Numerous studies have focused on single-walled carbon nanotubes (SWCNTs) and their ability to cause pulmonary injury related to fibrosis, and cancer; however few studies have addressed their impact on infectious agents, particularly viruses that are known for causing severe disease. Here we have demonstrated the ability of pristine SWCNTs of diverse electronic structure to increase the susceptibility of small airway epithelial cells (SAEC) to pandemic influenza A H1N1 infection and discerned potential mechanisms of action driving this response., Methods: Small airway epithelial cells (SAEC) were exposed to three types of SWCNTs with varying electronic structure (SG65, SG76, CG200) followed by infection with A/Mexico/4108/2009 (pH1N1). Cells were then assayed for viral infectivity by immunofluorescence and viral titers. We quantified mRNA and protein levels of targets involved in inflammation and anti-viral activity (INFβ1, IL-8, RANTES/CCL5, IFIT2, IFIT3, ST3GAL4, ST6GAL1, IL-10), localized sialic acid receptors, and assessed mitochondrial function. Hyperspectral imaging analysis was performed to map the SWCNTs and virus particles in fixed SAEC preparations. We additionally performed characterization analysis to monitor SWCNT aggregate size and structure under biological conditions using dynamic light scattering (DLS), static light scattering (SLS)., Results: Based on data from viral titer and immunofluorescence assays, we report that pre-treatment of SAEC with SWCNTs significantly enhances viral infectivity that is not dependent on SWCNT electronic structure and aggregate size within the range of 106 nm - 243 nm. We further provide evidence to support that this noted effect on infectivity is not likely due to direct interaction of the virus and nanoparticles, but rather a combination of suppression of pro-inflammatory (RANTES) and anti-viral (IFIT2, IFIT3) gene/protein expression, impaired mitochondrial function and modulation of viral receptors by SWCNTs., Conclusions: Results of this work reveal the potential for SWCNTs to increase susceptibility to viral infections as a mechanism of adverse effect. These data highlight the importance of investigating the ability of carbon-nanomaterials to modulate the immune system, including impacts on anti-viral mechanisms in lung cells, thereby increasing susceptibility to infectious agents.

The vitellogenin receptor (Vtgr) plays an important role in fish reproduction. This receptor functions to incorporate vitellogenin (Vtg), a macromolecule synthesized and released from the liver in the bloodstream, into oocytes where it is processed into yolk. Although studies have focused on the functional role of Vtgr in fish, the mechanistic control of this gene is still unexplored. Here we report the identification and analysis of the first piscine 5' regulatory region of the vtgr gene which was cloned from largemouth bass (Micropterus salmoides). Using this putative promoter sequence, we investigated a role for hormones, including insulin and 17β-estradiol (E2), in transcriptional regulation through cell-based reporter assays. No effect of insulin was observed, however, E2 was able to repress transcriptional activity of the vtgr promoter through select estrogen receptor subtypes, Esr1 and Esr2a but not Esr2b. Electrophoretic mobility shift assay demonstrated that Esr1 likely interacts with the vtgr promoter region through half ERE and/or SP1 sites, in part. Finally we also show that ethinylestradiol (EE2), but not bisphenol-A (BPA), represses promoter activity similarly to E2. These results reveal for the first time that the Esr1 isoform may play an inhibitory role in the expression of LMB vtgr mRNA under the influence of E2, and potent estrogens such as EE2. In addition, this new evidence suggests that vtgr may be a target of select endocrine disrupting compounds through environmental exposures., (© The Author 2014. Published by Oxford University Press on behalf of the Society of Toxicology. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Conjugation of multiple nanomaterials has become the focus of recent materials development. This new material class is commonly known as nanohybrids or "horizon nanomaterials". Conjugation of metal/metal oxides with carbonaceous nanomaterials and overcoating or doping of one metal with another have been pursued to enhance material performance and/or incorporate multifunctionality into nano-enabled devices and processes. Nanohybrids are already at use in commercialized energy, electronics and medical products, which warrant immediate attention for their safety evaluation. These conjugated ensembles likely present a new set of physicochemical properties that are unique to their individual component attributes, hence increasing uncertainty in their risk evaluation. Established toxicological testing strategies and enumerated underlying mechanisms will thus need to be re-evaluated for the assessment of these horizon materials. This review will present a critical discussion on the altered physicochemical properties of nanohybrids and analyze the validity of existing nanotoxicology data against these unique properties. The article will also propose strategies to evaluate the conjugate materials' safety to help undertake future toxicological research on the nanohybrid material class.

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.

Introduction: It has been hypothesized that racial disparities among several diseases are explained by differences in testosterone (T), 17-β estradiol (E), sex hormone binding globulin (SHBG) and albumin (A) levels, yet epidemiologic results have been mixed. Statistical advice regarding appropriate adjustment methods for carrier proteins of sex steroid hormones in the literature is scant. Therefore, we investigated different adjustment methods for carrier proteins., Methods: Data for 1496 men, >17 years from the Third National Health and Nutrition Examination Survey (NHANES III) 1988-91 were used to analyze linearity between sex hormones and carrier proteins by examining correlation, plots, and regression models. The statistical importance of age, body mass index (BMI), and race/ethnicity were examined for changes in results by the adjustment method., Results: T was weakly correlated with SHBG and A (r-squared, 0.25, 0.13, respectively) and E was weakly negatively correlated with A (p<0.0001), but not SHBG (p<0.1799). Based on the model residual plots and r-squared, the categorical model performed better than linear models. Regression coefficients for age, BMI, and race/ethnicity groups using quotient (e.g., T/A and E/A) models differed from continuous and categorical models., Conclusion: Choosing an appropriate adjustment for carrier proteins is important to prevent bias in analyses and inconsistency in findings across studies. Linearity between variables should not be assumed when adjusting models, and should be conducted and reported. An independent categorical carrier protein variable is recommended in analysis exploring factors predicting sex hormone levels, although statistical testing should always be employed., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Detection of SWCNTs in complex matrices presents a unique challenge as common techniques lack spatial resolution and specificity. Near infrared fluorescence (NIRF) has emerged as a valuable tool for detecting and quantifying SWCNTs in environmental samples by exploiting their innate fluorescent properties. The objective of this study was to optimize NIRF-based imaging and quantitation methods for tracking and quantifying SWCNTs in an aquatic vertebrate model in conjunction with assessing toxicological end points. Fathead minnows (Pimephales promelas) were exposed by single gavage to SWCNTs and their distribution was tracked using a custom NIRF imaging system for 7 days. No overt toxicity was observed in any of the SWCNT treated fish; however, histopathology observations from gastrointestinal (GI) tissue revealed edema within the submucosa and altered mucous cell morphology. NIRF images showed strong SWCNT-derived fluorescence signals in whole fish and excised intestinal tissues. Fluorescence was not detected in other tissues examined, indicating that no appreciable intestinal absorption occurred. SWCNTs were quantified in intestinal tissues using a NIRF spectroscopic method revealing values that were consistent with the pattern of fluorescence observed with NIRF imaging. Results of this work demonstrate the utility of NIRF imaging as a valuable tool for examining uptake and distribution of SWCNTs in aquatic vertebrates.

Aggregate structure of covalently functionalized chiral specific semiconducting single-walled carbon nanotubes (SWNTs) was systematically studied employing static light scattering (SLS). Fractal dimensions (Df) of two specific chirality SWNTs-SG65 and SG76 with (6, 5) and (7, 6) chiral enrichments-were measured under four biological exposure media conditions, namely: Dulbecco's Modified Eagle Medium (DMEM), Minimum Essential Medium (MEM), Roswell Park Memorial Institute (RPMI) 1640 medium, and 0.9% saline solution. The SWNTs exhibited chiral dependence on Df with SG65 showing more fractal or loosely bound aggregate structures, i.e., lower Df values (range of 2.24±0.03 to 2.64±0.05), compared to the SG76 sample (range of 2.58±0.13 to 2.90±0.08). All the Df values reported are highly reproducible, measured from multiple SLS runs and estimated with 'random block-effects' statistical analysis that yielded all p values to be <0.001. The key mechanism for such difference in Df between the SWNT samples was identified as the difference in van der Waals (VDW) interaction energies of these samples, where higher VDW of SG76 resulted in tighter packing density. Effect of medium type showed lower sensitivity; however, presence of di-valent cations (Ca(2+)) in DMEM and MEM media resulted in relatively loose or more fractal aggregates. Moreover, presence of fetal bovine serum (FBS) and bovine serum albumin (BSA), used to mimic the in vitro cell culture condition, reduced the Df values, i.e., created more fractal structures. Steric hindrance to aggregation was identified as the key mechanism for creating the fractal structures. Also, increase in FBS concentration from 1% to 10% resulted in increasingly lower Df values., (Published by Elsevier Ltd.)

Single-walled carbon nanotubes (SWNTs) are being used in many consumer products and devices. It is likely that as some of these products reach the end of their useful life, they will be discarded in municipal solid waste landfills. However, there has been little work evaluating the fate of nanomaterials in solid waste environments. The purpose of this study is to systematically evaluate the influence of organic matter type and concentration in landfill-relevant conditions on SWNT transport through a packed-bed of mixed municipal solid waste collectors. The influence of individual waste materials on SWNT deposition is also evaluated. Transport experiments were conducted through saturated waste-containing columns over a range of simulated leachate conditions representing both mature and young leachates. Results indicate that SWNT transport may be significant in mature waste environments, with mobility decreasing with decreasing humic acid concentration. SWNT mobility in the presence of acetic acid was inhibited, suggesting their mobility in young waste environments may be small. SWNTs also exhibited collector media-dependent transport, with greatest transport in glass and least in paper. These results represent the first study evaluating how leachate age and changes in waste composition influence potential SWNT mobility in landfills.

Aggregation kinetics of chiral-specific semiconducting single-walled carbon nanotubes (SWNTs) was systematically studied through time-resolved dynamic light scattering. Varied monovalent (NaCl) and divalent (CaCl(2)) electrolyte composition was used as background solution chemistry. Suwannee River humic acid (SRHA) was used to study the effects of natural organic matter on chirally separated SWNT aggregation. Increasing salt concentration and introduction of divalent cations caused aggregation of SWNT clusters by suppressing the electrostatic repulsive interaction from the oxidized surfaces. The (6,5) SWNTs, i.e., SG65, with relatively lower diameter tubes compared to (7,6), i.e., SG76, showed substantially higher stability (7- and 5-fold for NaCl and CaCl(2), respectively). The critical coagulation concentration (CCC) values were 96 and 13 mM NaCl in the case of NaCl and 2.8 and 0.6 mM CaCl(2) for SG65 and SG76, respectively. The increased tube diameter for (7,6) armchair SWNTs likely presented with higher van der Waals interaction and thus increased the aggregation propensity substantially. The presence of SRHA enhanced SWNT stability in divalent CaCl(2) environment through steric interaction from adsorbed humic molecules; however showed little or no effects for monovalent NaCl. The mechanism of aggregation-describing favorable interaction tendencies for (7,6) SWNTs-is probed through ab initio molecular modeling. The results suggest that SWNT stability can be chirality dependent in typical aquatic environment.

Objectives: Since sex hormone markers are metabolically linked, examining sex steroid hormones singly may account for inconsistent findings by age, race/ethnicity and body mass index (BMI) across studies. First, these markers were statistically combined into profiles to account for the metabolic relationship between markers. Then, the relationships between sex steroid hormone profiles and age, race/ethnicity and BMI were explored in multinomial logistic regression models., Design: Cross-sectional survey., Setting: The US Third National Health and Nutrition Examination Survey (NHANES III)., Participants: 1538 Men, >17 years., Primary Outcome Measure: Sex hormone profiles., Results: Cluster analysis was used to identify four statistically determined profiles with Blom-transformed T, E, sex hormone binding globulin (SHBG), and 3-α diol G. We used these four profiles with multinomial logistic regression models to examine differences by race/ethnicity, age and BMI. Mexican American men >50 years were associated with the profile that had lowest T, E and 3-α diol G levels compared to other profiles (p<0.05). Non-Hispanic Black, overweight (25-29.9 kg/m(2)) and obese (>30 kg/m(2)) men were most likely to be associated with the cluster with the lowest SHBG (p<0.05)., Conclusion: The associations of sex steroid hormone profiles by race/ethnicity are novel, while the findings by age and BMI groups are largely consistent with observations from single hormone studies. Future studies should validate these hormone profile groups and investigate these profiles in relation to chronic diseases and certain cancers.

Silver nanomaterials are the major components of healthcare products largely because of their antimicrobial effects. However, their unintended toxicity to biological organisms and its mechanism are not well understood. Using medaka fish embryo model, the toxic effects and corresponding mechanisms of silver nanocolloids (SNC, particle size 3.8 ± 1.0-diameter nm) were investigated. SNC caused morphological changes in embryos including cardiovascular malformations, ischemia, underdeveloped central nervous system and eyes, and kyphosis at exposures of 0.5 mg/L. Interestingly, SNC were observed inside the eggs at a level of 786.1 ± 32.5 pg/mg egg weight, and TEM analysis showed that SNC adhered to the surface and inside of the chorion. Meanwhile, medaka oligo DNA microarray and qRT-PCR were used for gene expression analysis in the embryos exposed to 0.05 mg/L SNC for 48 h. As a result, expressions of six of the oxidative stress-, embryogenesis- and morphogenesis-related genes, ctsL, tpm1, rbp, mt, atp2a1, and hox6b6, were affected by the SNC exposure, and these genes' involvement in those malformations was implied. Thus, SNC could potentially cause malformations in the cardiovascular and central nervous systems in developing medaka embryo through SNC-induced differential expression of the genes related to oxidative stress, embryonic cellular proliferation, and morphological development.

Understanding plant interactions with nanoparticles is of increasing importance for assessing their toxicity and trophic transport. The primary objective of this study was to assess uptake, biodistribution and toxicity associated with exposure of tobacco plants (Nicotiana xanthi) to gold nanoparticles (AuNPs). We employed synchrotron-based X-ray microanalysis with X-ray absorption near-edge microspectroscopy and high resolution electron microscopy to localize AuNPs within plants. Results from these experiments reveal that AuNPs entered plants through the roots and moved into the vasculature. Aggregate bodies were also detected within root cell cytoplasm. Furthermore, AuNP uptake was size selective as 3.5 nm AuNP spheres were detected in plants but 18 nm AuNPs remained agglomerated on the root outer surfaces. Finally, leaf necrosis was observed after 14 days of exposure to 3.5 nm AuNPs. Overall, results of this work show the potential for AuNPs to enter plants through size-dependent mechanisms, translocate to cells and tissues and cause biotoxicity.

Our laboratory has previously reported significant increases of the proinflammatory cytokine TNF-α in male hearts secondary to sustained volume overload. These elevated levels of TNF-α are accompanied by left ventricular (LV) dilatation and cardiac dysfunction. In contrast, estrogen has been shown to protect against this adverse cardiac remodeling in both female and male rats. The purpose of this study was to determine whether estrogen has an effect on inflammation-related genes that contribute to this estrogen-mediated cardioprotection. Myocardial volume overload was induced by aortocaval fistula in 8 wk old male Sprague-Dawley rats (n = 30), and genes of interest were identified using an inflammatory PCR array in Sham, Fistula, and Fistula + Estrogen-treated (0.02 mg/kg per day beginning 2 wk prior to fistula) groups. A total of 55 inflammatory genes were modified (≥2-fold change) at 3 days postfistula. The number of inflammatory gene was reduced to 21 genes by estrogen treatment, whereas 13 genes were comparably modulated in both fistula groups. The most notable were TNF-α, which was downregulated by estrogen, and the TNF-α receptors, which were differentially regulated by estrogen. Specific genes related to arachidonic acid metabolism were downregulated by estrogen, including cyclooxygenase-1 and -2. Finally, gene expression for the β1-integrin cell adhesion subunit was significantly upregulated in the LV of estrogen-treated animals. Protein levels reflected the changes observed at the gene level. These data suggest that estrogen provides its cardioprotective effects, at least in part, via genomic modulation of numerous inflammation-related genes.

The marine copepod, Amphiascus tenuiremis (A. tenuiremis), is a well characterized invertebrate model for the screening and evaluation of endocrine and reproductive toxins using life-cycle assays. These tests evaluate phenotypic endpoints related to development and reproduction, which are utilized to predict population outcomes. Some of these endpoints in arthropods, including sexual maturation and molting, are controlled by the hormone ecdysone which acts through its cognate receptor, the ecdysone receptor. The purpose of this research was to obtain and characterize sequence information for the A. tenuiremis ecdysone receptor and investigate modulation of expression levels by fipronil, an insecticide that causes infertility in males and reduced egg extrusion in female copepods, and ponasterone, a natural ecdysone receptor agonist. Results show successful cloning and phylogenetic analysis of the ecdysone receptor for A. tenuiremis, providing the first genetic information for a hormone receptor in this species. Exposure of copepodites to fipronil for 1, 2, 4, 18 and 30 h caused a significant increase in ecdysone receptor transcriptional expression at 30 h compared to control unexposed animals. This work illustrates a potential mechanism whereby exposure to fipronil, and potentially other endocrine disrupting compounds, results in impacted reproduction. Furthermore, this exemplifies the potential utility of ecdysone receptor transcriptional measurement as a sensitive and rapid biomarker of ecological relevance when linked to traditional A. tenuiremis bioassays., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Ecotoxicology research is using many methods for engineered nanomaterials (ENMs), but the collective experience from researchers has not been documented. This paper reports the practical issues for working with ENMs and suggests nano-specific modifications to protocols. The review considers generic practical issues, as well as specific issues for aquatic tests, marine grazers, soil organisms, and bioaccumulation studies. Current procedures for cleaning glassware are adequate, but electrodes are problematic. The maintenance of exposure concentration is challenging, but can be achieved with some ENMs. The need to characterize the media during experiments is identified, but rapid analytical methods are not available to do this. The use of sonication and natural/synthetic dispersants are discussed. Nano-specific biological endpoints may be developed for a tiered monitoring scheme to diagnose ENM exposure or effect. A case study of the algal growth test highlights many small deviations in current regulatory test protocols that are allowed (shaking, lighting, mixing methods), but these should be standardized for ENMs. Invertebrate (Daphnia) tests should account for mechanical toxicity of ENMs. Fish tests should consider semistatic exposure to minimize wastewater and animal husbandry. The inclusion of a benthic test is recommended for the base set of ecotoxicity tests with ENMs. The sensitivity of soil tests needs to be increased for ENMs and shortened for logistics reasons; improvements include using Caenorhabditis elegans, aquatic media, and metabolism endpoints in the plant growth tests. The existing bioaccumulation tests are conceptually flawed and require considerable modification, or a new test, to work for ENMs. Overall, most methodologies need some amendments, and recommendations are made to assist researchers., (Copyright © 2011 SETAC.)

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-1β, IL-4, IL-6, IL-12 subunit p40, MIP1α, MIP1β, 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-1β 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., (Copyright © 2011 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.)

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.

Bone marrow stromal cells (BMSCs) possess multi-lineage differentiation potential and can be induced to undergo differentiation into various cell types with the correct combination of chemical and environmental factors. Although, they have shown great prospects in therapeutic and medical applications, less is known about their behavior on nanosurfaces mimicking the extra cellular matrix (ECM). In this report we have employed 2D substrates coated with tobacco mosaic virus (TMV) nanorods to study the differentiation process of BMSCs into osteoblast like cells. TMV is a rod-shaped plant virus with an average length of 300 nm and diameter of 18 nm. The osteogenic differentiation of BMSCs on TMV was studied over time points of 7, 14 and 21 days. We examined the temporal gene expression changes during these time points by real-time quantitative PCR (RT-qPCR) analysis. As expected, osteo-specific genes (osteocalcin, osteopontin and osteonectin) were upregulated and showed a maximum change in expression on TMV at 14 days which was 7 days earlier than on tissue culture plastic (TCP). Based on the genes expression profile generated by RT-qPCR experiments, we proposed that the early interaction of cells with TMV triggers on signaling pathways which regulate speedy expression of osteocalcin in turn, resulting in early mineralization of the cells. To further investigate these regulating factors we studied global changes in gene expression (DNA microarray analyses) during osteogenic differentiation on the nanosubstrate. Multitudes of genes were affected by culturing cells on nanorod substrate, which corroborated our initial PCR findings. Microarray analysis further revealed additional targets influenced by the presence of nanorods on the surface, of which, the expression of bone morphogenetic protein 2 (BMP2) was of particular interests. Further investigation into the temporal change of BMP2, revealed that it acts as a major promoter in signaling the early regulation of osteocalcin on TMV coated substrates., ((c) 2009 Elsevier Ltd. All rights reserved.)

Fish embryo toxicology is important because embryos are considered more susceptible than adult fish to the effects of toxic chemicals. Recently, fish embryo bioassay was proposed to replace the conventional fish acute toxicity chemical test of the Organization for Economic Cooperation and Development guidelines because it offers the advantages of fewer reagents, easy handling, and efficient data production. To accelerate the establishment of a chemical toxicity database for the protection of environmental and human health, we need to determine whether the conventional toxicity test can safely be replaced by such fish embryo toxicity tests. For instance, it is unclear how the presence of the chorion moderates the toxic effects of some chemicals. If such chemical toxicities do differ between embryos and, for example, the larval stage, then different toxic effects should appear in later life. We tested the later-life effects of the neurotoxic insecticide carbaryl at sublethal concentrations (0 [control] and 5 and 10 mg/L) in embryos and posthatch larvae of the freshwater fish medaka, Oryzias latipes. Although embryos exposed until hatching showed multiple developmental malformations and reductions in subsequent survival rates over three months, no significant reduction was observed in tolerance to starvation for 7 d and in intrinsic population growth rate (r). Exposure of larvae for 96 h resulted in dose-responsive vertebral fracture, significant reduction in tolerance to starvation for 7 d, and reduced three-month survival rate; r was reduced significantly and consistently. These results suggested that posthatch larvae were more susceptible than embryos to carbaryl exposure and that the toxic cascades may differ between larvae and embryos. The influences of carbaryl exposure on population growth rate differed significantly with developmental stage.

The extracellular signal-regulated kinases 1 and 2 (ERK1/2) are phosphorylated after inhalation of asbestos. The effect of blocking this signaling pathway in lung epithelium is unclear. Asbestos-exposed transgenic mice expressing a dominant-negative mitogen-activated protein kinase kinase-1 (dnMEK1) (i.e., the upstream kinase necessary for phosphorylation of ERK1/2) targeted to lung epithelium exhibited morphologic and molecular changes in lung. Transgene-positive (Tg+) (i.e., dnMEK1) and transgene-negative (Tg-) littermates were exposed to crocidolite asbestos for 2, 4, 9, and 32 days or maintained in clean air (sham controls). Distal bronchiolar epithelium was isolated using laser capture microdissection and mRNA analyzed for molecular markers of proliferation and Clara cell secretory protein (CCSP). Lungs and bronchoalveolar lavage fluids were analyzed for inflammatory and proliferative changes and molecular markers of fibrogenesis. Distal bronchiolar epithelium of asbestos-exposed wild-type mice showed increased expression of c-fos at 2 days. Elevated mRNA levels of histone H3 and numbers of Ki-67-labeled proliferating bronchiolar epithelial cells were decreased at 4 days in asbestos-exposed Tg+ mice. At 32 days, distal bronchioles normally composed of Clara cells in asbestos-exposed Tg+ mouse lungs exhibited nonreplicating ciliated and mucin-secreting cells as well as decreased mRNA levels of CCSP. Gene expression (procollagen 3-a-1, procollagen 1-a-1, and IL-6) linked to fibrogenesis was also increased in lung homogenates of asbestos-exposed Tg- mice, but reduced in asbestos-exposed Tg+ mice. These results suggest a critical role of MEK1 signaling in epithelial cell proliferation and lung remodeling after toxic injury.

The ligand hepatocyte growth factor/scatter factor (HGF) and its receptor tyrosine kinase, c-Met, are highly expressed in most human malignant mesotheliomas (MMs) and may contribute to their increased growth and viability. Based upon our observation that RNA silencing of fos-related antigen 1 (Fra-1) inhibited c-met expression in rat mesotheliomas (1), we hypothesized that Fra-1 was a key player in HGF-induced proliferation in human MMs. In three of seven human MM lines evaluated, HGF increased Fra-1 levels and phosphorylation of both extracellular signal-regulated kinase 5 (ERK5) and AKT that were inhibited by the phosphatidylinositol 3-kinase (PI3K) inhibitor, LY290042. HGF-dependent phosphorylation and Fra-1 expression were decreased after knockdown of Fra-1, whereas overexpression of Fra-1 blocked the expression of mitogen/extracellular signal-regulated kinase kinases (MEK)5 at the mRNA and protein levels. Stable MM cell lines using a dnMEK5 showed that basal Fra-1 levels were increased in comparison to empty vector control lines. HGF also caused increased MM cell viability and proliferating cell nuclear antigen (PCNA) expression that were abolished by knockdown of MEK5 or Fra-1. Data suggest that HGF-induced effects in some MM cells are mediated via activation of a novel PI3K/ERK5/Fra-1 feedback pathway that might explain tumor-specific effects of c-Met inhibitors on MM and other tumors.

The estrogen receptor (ER) signaling cascade is a vulnerable target of exposure to environmental xenoestrogens, like nonylphenol (NP), which are causally associated with impaired health status. However, the impact of xenoestrogens on the individual receptor isotypes (alpha, beta a, and beta b) is not well understood. The goal of these studies was to determine the impact of NP on largemouth bass (Micropterus salmoides) ER isotype expression and activity. Here, we show that hepatic expression levels of three receptors are not equivalent in male largemouth bass exposed to NP by injection. Transcript levels of the ER alpha subtype were predominantly induced in concert with vitellogenin similarly to fish exposed to 17beta-estradiol (E(2)) as measured by quantitative real-time PCR. NP also induced circulating plasma levels of estrogen, which may contribute to overall activation of the ERs. To measure the activation of each receptor isotype by E(2) and NP, we employed reporter assays using an estrogen response element (ERE)-luciferase construct. Results from these studies show that ER alpha had the greatest activity following exposure to E(2) and NP. This activity was inhibited by the antagonists ICI 182 780 and ZM 189 154. Furthermore, both beta b and beta a subtypes depressed ER alpha activation, suggesting that the cellular composition of receptor isotypes may contribute to the overall actions of estrogen and estrogenic contaminants via the receptors. Results from these studies collectively reveal the differential response of fish ER isotypes in response to xenoestrogens.

To elucidate genes important in development or repair of asbestos-induced lung diseases, gene expression was examined in mice after inhalation of chrysotile asbestos for 3, 9, and 40 days. We identified changes in the expression of genes linked to proliferation (cyclin B2, CDC20, and CDC28 protein kinase regulatory subunit 2), inflammation (CCL9, CCL6, complement component 1, chitinase3-like 3, TNF superfamily member 10, and IL-1B), and matrix remodeling (MMP12, MMP3, integrin alphaX, and cathepsins K, Z, B, and S). The most highly induced gene at all time points was mclca3 (gob5), a putative calcium-activated chloride channel involved in the regulation of mucus production and/or secretion. Using histochemistry, we demonstrated accumulation of mucus and increased mClca3 protein in the bronchiolar epithelium of asbestos-exposed mice at all time points but peaking at 9 days. Cytokine levels (interleukin-1beta, interleukin-4, interleukin-6) in bronchoalveolar lavage fluid also increased at 9 days, suggesting Th2-mediated immunity may play a role in asbestos-induced mucus production. In contrast, levels of cathepsin K, a potent elastase, increased between 3 and 40 days at both the mRNA and protein levels, localizing primarily in CD45-positive leukocytes and interstitial cells. Identification of genes involved in lung injury and remodeling after asbestos exposure could aid in defining mechanisms of airborne particulate-induced disease and in developing therapeutic strategies.

Malignant mesothelioma is an aggressive cancer with no known cure, which has become a therapeutic challenge. Onconase is one of few chemotherapeutic agents that have been studied in patients with malignant mesothelioma that has the advantage of low toxicity and limited side effects. Here, we evaluate the effect of Onconase on killing of malignant mesothelioma cells and how the phosphatidylinositol 3-kinase/AKT (PI3-K/AKT) survival pathway influences this effect. Our results show that Onconase induces apoptosis in malignant mesothelioma cell lines and that this effect is tumor cell specific. Malignant mesothelioma cell lines with the highest AKT activation, which correlated with the presence of the SV40 large and small T antigen (SV40+), were the most resistant to the drug. Finally, a cooperative effect was observed between small molecule inhibitors of PI3-K and Onconase in the killing of malignant mesothelioma cells. Our results suggest that kinase screening of individual malignant mesotheliomas for endogenous levels of activated PI3-K/AKT may be predictive of the efficacy of Onconase and possibly other chemotherapeutic agents.

The expression levels of three estrogen receptor (ER) isotypes alpha, beta, and gamma were quantified in female largemouth bass (Micropterus salmoides) (LMB) liver, ovary, brain, and pituitary tissues. ER alpha and beta expression predominated in the liver, while ERs beta and gamma predominated in the other tissues. Temporally in females, ER alpha was highly up-regulated, ER gamma was slightly up-regulated, and ER beta levels remained unchanged in the liver when plasma 17-beta estradiol (E2) and vitellogenin (Vtg) levels were elevated in the spring. In ovarian tissue from these same fish, all three ERs were maximally expressed in the fall, during early oocyte development and prior to peak plasma E2 levels. When males were injected with E2, ER alpha was highly inducible, ER gamma was moderately up-regulated, and ER beta levels were not affected. None of the ER isotypes were induced by E2 in gonadal tissues. These results combined suggest that the ERs themselves are not regulated in the same manner by E2, and furthermore, do not contribute equally to the transcriptional regulation of genes involved in fish reproduction such as Vtg.