Crystalline silica-induced pulmonary inflammation and autoimmunity in mature adult NZBW/f1 mice: age-related sensitivity and impact of omega-3 fatty acid intervention.

OBJECTIVE: Occupational exposure to respirable crystalline silica (cSiO2) has been linked to lupus development. Previous studies in young lupus-prone mice revealed that intranasal cSiO2 exposure triggered autoimmunity, preventable with docosahexaenoic acid (DHA). This study explores cSiO2 and DHA effects in mature lupus-prone adult mice, more representative of cSiO2-exposed worker age. METHODS: Female NZBWF1 mice (14-week old) were fed control (CON) or DHA-supplemented diets. After two weeks, mice were intranasally instilled saline (VEH) or 1 mg cSiO2 weekly for four weeks. Cohorts were then analyzed 1- and 5-weeks postinstillation for lung inflammation, cell counts, chemokines, histopathology, B- and T-cell infiltration, autoantibodies, and gene signatures, with results correlated to autoimmune glomerulonephritis onset. RESULTS: VEH/CON mice showed no pathology. cSiO2/CON mice displayed significant ectopic lymphoid tissue formation in lungs at 1 week, increasing by 5 weeks. cSiO2/CON lungs exhibited elevated cellularity, chemokines, CD3+ T-cells, CD45R + B-cells, IgG + plasma cells, gene expression, IgG autoantibodies, and glomerular hypertrophy. DHA supplementation mitigated all these effects. DISCUSSION: The mature adult NZBWF1 mouse used here represents a life-stage coincident with immunological tolerance breach and one that more appropriately represents the age (20-30 yr) of cSiO2-exposed workers. cSiO2-induced robust pulmonary inflammation, autoantibody responses, and glomerulonephritis in mature adult mice, surpassing effects observed previously in young adults. DHA at a human-equivalent dosage effectively countered cSiO2-induced inflammation/autoimmunity in mature mice, mirroring protective effects in young mice. CONCLUSION: These results highlight life-stage significance in this preclinical lupus model and underscore omega-3 fatty acids' therapeutic potential against toxicant-triggered autoimmune responses.

Comparative mechanisms of PAH toxicity by benzo[a]pyrene and dibenzo[def,p]chrysene in primary human bronchial epithelial cells cultured at air-liquid interface.

Current assumption for assessing carcinogenic risk of polycyclic aromatic hydrocarbons (PAHs) is that they function through a common mechanism of action; however, recent studies demonstrate that PAHs can act through unique mechanisms potentially contributing to cancer outcomes in a non-additive manner. Using a primary human 3D bronchial epithelial culture (HBEC) model, we assessed potential differences in mechanism of toxicity for two PAHs, benzo[a]pyrene (BAP) and dibenzo[def,p]chrysene (DBC), compared to a complex PAH mixture based on short-term biosignatures identified from transcriptional profiling. Differentiated bronchial epithelial cells were treated with BAP (100-500 µg/ml), DBC (10 µg/ml), and coal tar extract (CTE 500-1500 µg/ml, SRM1597a) for 48 h and gene expression was measured by RNA sequencing or quantitative PCR. Comparison of BAP and DBC gene signatures showed that the majority of genes (~60%) were uniquely regulated by treatment, including signaling pathways for inflammation and DNA damage by DBC and processes for cell cycle, hypoxia and oxidative stress by BAP. Specifically, BAP upregulated targets of AhR, NRF2, and KLF4, while DBC downregulated these same targets, suggesting a chemical-specific pattern in transcriptional regulation involved in antioxidant response, potentially contributing to differences in PAH potency. Other processes were regulated in common by all PAH treatments, BAP, DBC and CTE, including downregulation of genes involved in cell adhesion and reduced functional measurements of barrier integrity. This work supports prior in vivo studies and demonstrates the utility of profiling short-term biosignatures in an organotypic 3D model to identify mechanisms linked to carcinogenic risk of PAHs in humans.

Use of a modified GreenScreen tool to conduct a screening-level comparative hazard assessment of conventional silver and two forms of nanosilver.

BACKGROUND: Increased concern for potential health and environmental impacts of chemicals, including nanomaterials, in consumer products is driving demand for greater transparency regarding potential risks. Chemical hazard assessment is a powerful tool to inform product design, development and procurement and has been integrated into alternative assessment frameworks. The extent to which assessment methods originally designed for conventionally-sized materials can be used for nanomaterials, which have size-dependent physical and chemical properties, have not been well established. We contracted with a certified GreenScreen profiler to conduct three GreenScreen hazard assessments, for conventional silver and two forms of nanosilver. The contractor summarized publicly available literature, and used defined GreenScreen hazard criteria and expert judgment to assign and report hazard classification levels, along with indications of confidence in those assignments. Where data were not available, a data gap (DG) was assigned. Using the individual endpoint scores, an aggregated benchmark score (BM) was applied. RESULTS: Conventional silver and low-soluble nanosilver were assigned the highest possible hazard score and a silica-silver nanocomposite called AGS-20 could not be scored due to data gaps. AGS-20 is approved for use as antimicrobials by the US Environmental Protection Agency. CONCLUSIONS: An existing method for chemical hazard assessment and communication can be used - with minor adaptations- to compare hazards across conventional and nano forms of a substance. The differences in data gaps and in hazard profiles support the argument that each silver form should be considered unique and subjected to hazard assessment to inform regulatory decisions and decisions about product design and development. A critical limitation of hazard assessments for nanomaterials is the lack of nano-specific hazard data - where data are available, we demonstrate that existing hazard assessment systems can work. The work is relevant for risk assessors and regulators. We recommend that regulatory agencies and others require more robust data sets on each novel nanomaterial before granting market approval.

Subchronic intranasal lipopolysaccharide exposure induces pulmonary autoimmunity and glomerulonephritis in NZBWF1 mice.

Lupus, a systemic autoimmune disease shaped by gene-environment interplay, often progresses to endstage renal failure. While subchronic systemic exposure to bacterial lipopolysaccharide (LPS) triggers autoimmunity and glomerulonephritis in lupus-prone mice, it is unknown if inhaling LPS, which is common in certain occupations, can similarly trigger lupus. Here we determined how subchronic intranasal (IN) LPS instillation influences autoimmunity and glomerulonephritis development in lupusprone NZBWF1 female mice. Briefly, mice were IN-instilled with vehicle or E. coli LPS (0.8 µg/g) twice weekly for 5 wk, followed by necropsy. For systemic comparison, additional cohorts of mice were injected with LPS intraperitoneally (IP) using identical doses/timing. Lungs were assessed for inflammatory and autoimmune responses and then related to systemic autoimmunity and glomerulonephritis. IN/LPS exposure induced in the lung: i) leukocyte infiltration, ii)mRNA signatures for cytokines, chemokines, IFN-regulated, and cell death-related genes, iii) ectopic lymphoid tissue formation, and iv)diverse IgM and IgG autoantibodies (AAbs). Pulmonary effects coincided with enlarged spleens, elevated plasma IgG AAbs, and inflamed IgG-containing kidney glomeruli. In contrast, IP/LPS treatment induced systemic autoimmunity and glomerulonephritis without pulmonary manifestations. Taken together, these preclinical findings suggest the lung could serve as a critical nexus for triggering autoimmunity by respirable LPS in genetically predisposed individuals.

Lipidome modulation by dietary omega-3 polyunsaturated fatty acid supplementation or selective soluble epoxide hydrolase inhibition suppresses rough LPS-accelerated glomerulonephritis in lupus-prone mice.

Introduction: Lipopolysaccharide (LPS)-accelerated autoimmune glomerulonephritis (GN) in NZBWF1 mice is a preclinical model potentially applicable for investigating lipidome-modulating interventions against lupus. LPS can be expressed as one of two chemotypes: smooth LPS (S-LPS) or rough LPS (R-LPS) which is devoid of O-antigen polysaccharide sidechain. Since these chemotypes differentially affect toll-like receptor 4 (TLR4)-mediated immune cell responses, these differences may influence GN induction. Methods: We initially compared the effects of subchronic intraperitoneal (i.p.) injection for 5 wk with 1) Salmonella S-LPS, 2) Salmonella R-LPS, or 3) saline vehicle (VEH) (Study 1) in female NZBWF1 mice. Based on the efficacy of R-LPS in inducing GN, we next used it to compare the impact of two lipidome-modulating interventions, ω-3 polyunsaturated fatty acid (PUFA) supplementation and soluble epoxide hydrolase (sEH) inhibition, on GN (Study 2). Specifically, effects of consuming ω-3 docosahexaenoic acid (DHA) (10 g/kg diet) and/or the sEH inhibitor 1-(4-trifluoro-methoxy-phenyl)-3-(1-propionylpiperidin-4-yl) urea (TPPU) (22.5 mg/kg diet ≈ 3 mg/kg/day) on R-LPS triggering were compared. Results: In Study 1, R-LPS induced robust elevations in blood urea nitrogen, proteinuria, and hematuria that were not evident in VEH- or S-LPS-treated mice. R-LPS-treated mice further exhibited kidney histopathology including robust hypertrophy, hyperplasia, thickened membranes, lymphocytic accumulation containing B and T cells, and glomerular IgG deposition consistent with GN that was not evident in VEH- or SLPS-treated groups. R-LPS but not S-LPS induced spleen enlargement with lymphoid hyperplasia and inflammatory cell recruitment in the liver. In Study 2, resultant blood fatty acid profiles and epoxy fatty acid concentrations reflected the anticipated DHA- and TPPU-mediated lipidome changes, respectively. The relative rank order of R-LPS-induced GN severity among groups fed experimental diets based on proteinuria, hematuria, histopathologic scoring, and glomerular IgG deposition was: VEH/CON< R-LPS/DHA ≈ R-LPS/TPPU<<< R-LPS/TPPU+DHA ≈ R-LPS/CON. In contrast, these interventions had modest-to- negligible effects on R-LPS-induced splenomegaly, plasma antibody responses, liver inflammation, and inflammation-associated kidney gene expression. Discussion: We show for the first time that absence of O-antigenic polysaccharide in R-LPS is critical to accelerated GN in lupus-prone mice. Furthermore, intervention by lipidome modulation through DHA feeding or sEH inhibition suppressed R-LPS-induced GN; however, these ameliorative effects were greatly diminished upon combining the treatments.

Consecutive treatments of methamphetamine promote the development of cardiac pathological symptoms in zebrafish.

Chronic methamphetamine use, a widespread drug epidemic, has been associated with cardiac morphological and electrical remodeling, leading to the development of numerous cardiovascular diseases. While methamphetamine has been documented to induce arrhythmia, most results originate from clinical trials from users who experienced different durations of methamphetamine abuse, providing no documentation on the use of methamphetamine in standardized settings. Additionally, the underlying molecular mechanism on how methamphetamine affects the cardiovascular system remains elusive. A relationship was sought between cardiotoxicity and arrhythmia with associated methamphetamine abuse in zebrafish to identify and to understand the adverse cardiac symptoms associated with methamphetamine. Zebrafish were first treated with methamphetamine 3 times a week over a 2-week duration. Immediately after treatment, zebrafish underwent electrocardiogram (ECG) measurement using an in-house developed acquisition system for electrophysiological analysis. Subsequent analyses of cAMP expression and Ca2+ regulation in zebrafish cardiomyocytes were conducted. cAMP is vital to development of myocardial fibrosis and arrhythmia, prominent symptoms in the development of cardiovascular diseases. Ca2+ dysregulation is also a factor in inducing arrhythmias. During the first week of treatment, zebrafish that were administered with methamphetamine displayed a decrease in heart rate, which persisted throughout the second week and remained significantly lower than the heart rate of untreated fish. Results also indicate an increased heart rate variability during the early stage of treatment followed by a decrease in the late stage for methamphetamine-treated fish over the duration of the experiment, suggesting a biphasic response to methamphetamine exposure. Methamphetamine-treated fish also exhibited reduced QTc intervals throughout the experiment. Results from the cAMP and Ca2+ assays demonstrate that cAMP was upregulated and Ca2+ was dysregulated in response to methamphetamine treatment. Collagenic assays indicated significant fibrotic response to methamphetamine treatment. These results provide potential insight into the role of methamphetamine in the development of fibrosis and arrhythmia due to downstream effectors of cAMP.

Comparative effects of human-equivalent low, moderate, and high dose oral prednisone intake on autoimmunity and glucocorticoid-related toxicity in a murine model of environmental-triggered lupus.

Autoimmune diseases can be triggered by environmental toxicants such as crystalline silica dust (cSiO2). Here, we characterized the dose-dependent immunomodulation and toxicity of the glucocorticoid (GC) prednisone in a preclinical model that emulates onset and progression of cSiO2-triggered lupus. Two cohorts of 6-wk-old female NZBWF1 mice were fed either control AIN-93G diet or one of three AIN-93G diets containing prednisone at 5, 15, or 50 mg/kg diet which span human equivalent oral doses (HED) currently considered to be low (PL; 5 mg/d HED), moderate (PM; 14 mg/d HED), or high (PH; 46 mg/d HED), respectively. At 8 wk of age, mice were intranasally instilled with either saline vehicle or 1 mg cSiO2 once weekly for 4 wk. The experimental plan was to 1) terminate one cohort of mice (n=8/group) 14 wk after the last cSiO2 instillation for pathology and autoimmunity assessment and 2) to maintain a second cohort (n=9/group) to monitor glomerulonephritis development and survival. Mean blood concentrations of prednisone's principal active metabolite, prednisolone, in mice fed PL, PM, and PH diets were 27, 105, 151 ng/ml, respectively, which are consistent with levels observed in human blood ≤ 12 h after single bolus treatments with equivalent prednisone doses. Results from the first cohort revealed that consumption of PM, but not PL diet, significantly reduced cSiO2-induced pulmonary ectopic lymphoid structure formation, nuclear-specific AAb production, inflammation/autoimmune gene expression in the lung and kidney, splenomegaly, and glomerulonephritis in the kidney. Relative to GC-associated toxicity, PM diet, but not PL diet, elicited muscle wasting, but these diets did not affect bone density or cause glucosuria. Importantly, neither PM nor PL diet improved latency of cSiO2-accelerated death. PH-fed mice in both cohorts displayed robust GC-associated toxicity including body weight loss, reduced muscle mass, and extensive glucosuria 7 wk after the final cSiO2 instillation requiring their early removal from the study. Taken together, our results demonstrate that while moderate doses of prednisone can reduce important pathological endpoints of cSiO2-induced autoimmunity in lupus-prone mice, such as upstream ectopic lymphoid structure formation, these ameliorative effects come with unwanted GC toxicity, and, crucially, none of these three doses extended survival time.

Continuous Electrocardiogram Monitoring in Zebrafish with Prolonged Mild Anesthesia.

The zebrafish model has been demonstrated as an ideal vertebrate model system for a diverse range of biological studies. Along with conventional approaches, monitoring and analysis of zebrafish electrocardiogram (ECG) have been utilized for cardio-physiological screening and elucidation. ECG monitoring has been carried out with fish treated with anesthetic drugs, rendering the short period of time in recording the signals (<5 min). In this work, a prolonged sedation system for continuous ECG monitoring of multiple zebrafish was proposed and developed. We built a circulation system to provide prolonged mild anesthesia which allows more consistent and intrinsic ECG measurement. The use of prolonged anesthesia helped reduce the concentration of the anesthetic drug (MS222 or Tricaine) from 200 mg/L to 100 mg/L and even lower; thus, maintaining the integrity of intrinsic ECG. Moreover, heartrate variation during recording was investigated, showing minute changes (±3.2 beats per minute - BPM). The development of this prolonged ECG monitoring system would open the possibility of long-term monitoring for studies such as drug screening and forward genetic screening.

Promising Practices for Alternatives Assessment: Lessons from a Case Study of Copper-Free Antifouling Coatings.

Alternatives assessment (AA) is intended to identify safer and more sustainable approaches for managing chemicals used in industrial applications and consumer products and to avoid the adoption of regrettable substitutions. In the United States, the state of Washington prescribes a science-based approach for conducting an AA that meets regulatory requirements. This paper provides an overview of the approach, based on the Interstate Chemicals Clearinghouse (IC2) AA Guide, and illustrates its application to the examination of suitable alternatives to Cu-based antifouling coatings commonly used for recreational boats in the Pacific Northwest. Legislation has been passed in Washington State that will ban the use of certain Cu-based products in both freshwater and marine environments. The AA approach was used to identify and evaluate several alternatives to Cu-based antifouling boat paint products. Five promising practices that AA practitioners should consider when using the IC2 AA Guide in similar assessments of alternatives to industrial practices and consumer products include actively engaging stakeholders, enhancing the decision framework using a selection guide approach, scoping alternatives broadly, optimizing ingredient transparency, and identifying data gaps that could interfere with substitution efforts. The role AA plays in driving consumer product and similar technology innovations and its implications for the future are discussed. Environ Assess Manag 2019;00:1-13. © 2019 SETAC.

Lessons from the 2018 International Symposium on Alternatives Assessment: Advances and Reflections on Practice and Ongoing Needs to Build the Field.

Alternatives assessment is gaining traction as a systematic method to support the informed substitution of chemicals of concern. The 2nd International Symposium on Alternatives Assessment, on 1-2 November 2018, convened nearly 150 professionals from government agencies, industry, consultant firms, academia, and advocacy organizations to advance a greater understanding of the evolving methods, practices, and challenges in the use of alternatives assessment. This article reviews highlights and lessons from the symposium, including 1) notable advances in methods, 2) shared insights from practitioners on best practices as well as inherent tensions and challenges, and 3) research and practice needs in the field that can be addressed by organizations such as the newly launched Association for the Advancement of Alternatives Assessment. Being interdisciplinary in nature, the establishment of educational frameworks across disciplines and inclusion of diverse expertise in hazard and exposure assessments, life cycle impacts considerations, design principles, and economic and engineering evaluations will ensure continued growth of the field. Integr Environ Assess Manag 2019;00:1-8. © 2019 SETAC.

Cardiac tissue engineering: state-of-the-art methods and outlook.

The purpose of this review is to assess the state-of-the-art fabrication methods, advances in genome editing, and the use of machine learning to shape the prospective growth in cardiac tissue engineering. Those interdisciplinary emerging innovations would move forward basic research in this field and their clinical applications. The long-entrenched challenges in this field could be addressed by novel 3-dimensional (3D) scaffold substrates for cardiomyocyte (CM) growth and maturation. Stem cell-based therapy through genome editing techniques can repair gene mutation, control better maturation of CMs or even reveal its molecular clock. Finally, machine learning and precision control for improvements of the construct fabrication process and optimization in tissue-specific clonal selections with an outlook of cardiac tissue engineering are also presented.

Qualitative approach to comparative exposure in alternatives assessment.

Most alternatives assessments (AAs) published to date are largely hazard-based rankings, thereby ignoring potential differences in human and/or ecosystem exposures; as such, they may not represent a fully informed consideration of the advantages and disadvantages of possible alternatives. Building on the 2014 US National Academy of Sciences recommendations to improve AA decisions by including comparative exposure assessment into AAs, the Health and Environmental Sciences Institute's (HESI) Sustainable Chemical Alternatives Technical Committee, which comprises scientists from academia, industry, government, and nonprofit organizations, developed a qualitative comparative exposure approach. Conducting such a comparison can screen for alternatives that are expected to have a higher or different routes of human or environmental exposure potential, which together with consideration of the hazard assessment, could trigger a higher tiered, more quantitative exposure assessment on the alternatives being considered, minimizing the likelihood of regrettable substitution. This article outlines an approach for including chemical ingredient- and product-related exposure information in a qualitative comparison, including ingredient and product-related parameters. A classification approach was developed for ingredient and product parameters to support comparisons between alternatives as well as a methodology to address exposure parameter relevance and data quality. The ingredient parameters include a range of physicochemical properties that can impact routes and magnitude of exposure, whereas the product parameters include aspects such as product-specific exposure pathways, use information, accessibility, and disposal. Two case studies are used to demonstrate the application of the methodology. Key learnings and future research needs are summarized. Integr Environ Assess Manag 2018;00:000-000. © 2018 The Authors. Integrated Environmental Assessment and Management published by Wiley Periodicals, Inc. on behalf of Society of Environmental Toxicology & Chemistry (SETAC).

Advancing alternatives analysis: The role of predictive toxicology in selecting safer chemical products and processes.

Alternatives analysis (AA) is a method used in regulation and product design to identify, assess, and evaluate the safety and viability of potential substitutes for hazardous chemicals. It requires toxicological data for the existing chemical and potential alternatives. Predictive toxicology uses in silico and in vitro approaches, computational models, and other tools to expedite toxicological data generation in a more cost-effective manner than traditional approaches. The present article briefly reviews the challenges associated with using predictive toxicology in regulatory AA, then presents 4 recommendations for its advancement. It recommends using case studies to advance the integration of predictive toxicology into AA, adopting a stepwise process to employing predictive toxicology in AA beginning with prioritization of chemicals of concern, leveraging existing resources to advance the integration of predictive toxicology into the practice of AA, and supporting transdisciplinary efforts. The further incorporation of predictive toxicology into AA would advance the ability of companies and regulators to select alternatives to harmful ingredients, and potentially increase the use of predictive toxicology in regulation more broadly. Integr Environ Assess Manag 2017;13:915-925. © 2017 SETAC.