Hepatic Transcriptomic Patterns in the Neonatal Rat After Pentabromodiphenyl Ether Exposure.

Human exposure to pentabromodiphenyl ether (PBDE) mixture (DE-71) and its PBDE-47 congener can occur both in utero and during lactation. Here, we tested the hypothesis that PBDE-induced neonatal hepatic transcriptomic alterations in Wistar Han rat pups can inform on potential toxicity and carcinogenicity after longer term PBDE exposures. Wistar Han rat dams were exposed to either DE-71 or PBDE-47 daily from gestation day (GD 6) through postnatal day 4 (PND 4). Total plasma thyroxine (T4) was decreased in PND 4 pups. In liver, transcripts for CYPs and conjugation enzymes, Nrf2, and ABC transporters were upregulated. In general, the hepatic transcriptomic alterations after exposure to DE-71 or PBDE-47 were similar and provided early indicators of oxidative stress and metabolic alterations, key characteristics of toxicity processes. The transcriptional benchmark dose lower confidence limits of the most sensitive biological processes were lower for PBDE-47 than for the PBDE mixture. Neonatal rat liver transcriptomic data provide early indicators on molecular pathway alterations that may lead to toxicity and/or carcinogenicity if the exposures continue for longer durations. These early toxicogenomic indicators may be used to help prioritize chemicals for a more complete toxicity and cancer risk evaluation.

Sulforaphane enriched transcriptome of lung mitochondrial energy metabolism and provided pulmonary injury protection via Nrf2 in mice.

Nrf2 is essential to antioxidant response element (ARE)-mediated host defense. Sulforaphane (SFN) is a phytochemical antioxidant known to affect multiple cellular targets including Nrf2-ARE pathway in chemoprevention. However, the role of SFN in non-malignant airway disorders remain unclear. To test if pre-activation of Nrf2-ARE signaling protects lungs from oxidant-induced acute injury, wild-type (Nrf2+/+) and Nrf2-deficient (Nrf2-/-) mice were given SFN orally or as standardized broccoli sprout extract diet (SBE) before hyperoxia or air exposure. Hyperoxia-induced pulmonary injury and oxidation indices were significantly reduced by SFN or SBE in Nrf2+/+ mice but not in Nrf2-/- mice. SFN upregulated a large cluster of basal lung genes that are involved in mitochondrial oxidative phosphorylation, energy metabolism, and cardiovascular protection only in Nrf2+/+ mice. Bioinformatic analysis elucidated ARE-like motifs on these genes. Transcript abundance of the mitochondrial machinery genes remained significantly higher after hyperoxia exposure in SFN-treated Nrf2+/+ mice than in SFN-treated Nrf2-/- mice. Nuclear factor-κB was suggested to be a central molecule in transcriptome networks affected by SFN. Minor improvement of hyperoxia-caused lung histopathology and neutrophilia by SFN in Nrf2-/- mice implies Nrf2-independent or alternate effector mechanisms. In conclusion, SFN is suggested to be as a preventive intervention in a preclinical model of acute lung injury by linking mitochondria and Nrf2. Administration of SFN alleviated acute lung injury-like pathogenesis in a Nrf2-dependent manner. Potential AREs in the SFN-inducible transcriptome for mitochondria bioenergetics provided a new insight into the downstream mechanisms of Nrf2-mediated pulmonary protection.

Dosage compensation and DNA methylation landscape of the X chromosome in mouse liver.

DNA methylation plays a key role in X-chromosome inactivation (XCI), a process that achieves dosage compensation for X-encoded gene products between mammalian female and male cells. However, differential sex chromosome dosage complicates genome-wide epigenomic assessments, and the X chromosome is frequently excluded from female-to-male comparative analyses. Using the X chromosome in the sexually dimorphic mouse liver as a model, we provide a general framework for comparing base-resolution DNA methylation patterns across samples that have different chromosome numbers and ask at a systematic level if predictions by historical analyses of X-linked DNA methylation hold true at a base-resolution chromosome-wide level. We demonstrate that sex-specific methylation patterns on the X chromosome largely reflect the effects of XCI. While our observations concur with longstanding observations of XCI at promoter-proximal CpG islands, we provide evidence that sex-specific DNA methylation differences are not limited to CpG island boundaries. Moreover, these data support a model in which maintenance of CpG islands in the inactive state does not require complete regional methylation. Further, we validate an intragenic non-CpG methylation signature in genes escaping XCI in mouse liver. Our analyses provide insight into underlying methylation patterns that should be considered when assessing sex differences in genome-wide methylation analyses.

Erratum: "Diversity Outbred Mice Identify Population-Based Exposure Thresholds and Genetic Factors that Influence Benzene-Induced Genotoxicity".

[This corrects the article DOI: http://dx.doi.org/10.1289/ehp.1408202.].

Airway injury in an in vitro human epithelium-fibroblast model of diacetyl vapor exposure: diacetyl-induced basal/suprabasal spongiosis.

Inhalation exposure to diacetyl (DA) is associated with obliterative bronchiolitis (OB) in workers and induces OB-like fibrotic airway lesions in rats. The pathogenesis of OB is poorly understood in part due to complex interactions between airway epithelial, mesenchymal and blood-derived inflammatory cells. DA-induced airway toxicity in the absence of recruited-inflammatory/immune cells was characterized using an air-liquid interface (ALI) model consisting of human airway epithelium with (Epi/FT) and without (Epi) a mesenchymal component. ALI cultures were exposed to 25 mM DA-derived vapors (using vapor cups) for 1 h on day 0, 2 and 4. In some experiments, the tissues were exposed to 2,3-hexanedione (Hex) which is structurally-similar, but much less fibrogenic than DA. Lactate dehydrogenase activity and day 6 histopathologic changes associated with epithelial injury, including basal/suprabasal spongiosis, were increased following exposure of Epi/FT tissues to DA but not control or Hex vapors. IL-1a, IL-6, IL-8, sIL-1Ra, TGFa, MCP-3 and TNFa proteins were increased following DA exposure of Epi/FT tissues; only IL-1a, IL-8, sIL-1Ra and TGFa were increased following exposure of Epi tissues. MMP-1, MMP-3 and TIMP-1 proteins were increased following DA exposure of Epi/FT tissues; whereas MMP-2, MMP-7 and TIMP-2 were decreased, and production was largely dependent upon the presence of sub-epithelial stromal matrix/fibroblasts. Hex-induced protein changes were minimal. This in vitro study demonstrated that exposure of human airways to DA vapors induced epithelial injury (with the histopathologic feature of basal/suprabasal spongiosis) and increased release of pro-inflammatory and pro-fibrotic cytokines/chemokines as well as MMPs/TIMPs in the absence of recruited-inflammatory cells.

The Diacetyl-Exposed Human Airway Epithelial Secretome: New Insights into Flavoring-Induced Airways Disease.

Bronchiolitis obliterans (BO) is an increasingly important lung disease characterized by fibroproliferative airway lesions and decrements in lung function. Occupational exposure to the artificial food flavoring ingredient diacetyl, commonly used to impart a buttery flavor to microwave popcorn, has been associated with BO development. In the occupational setting, diacetyl vapor is first encountered by the airway epithelium. To better understand the effects of diacetyl vapor on the airway epithelium, we used an unbiased proteomic approach to characterize both the apical and basolateral secretomes of air-liquid interface cultures of primary human airway epithelial cells from four unique donors after exposure to an occupationally relevant concentration (∼1,100 ppm) of diacetyl vapor or phosphate-buffered saline as a control on alternating days. Basolateral and apical supernatants collected 48 h after the third exposure were analyzed using one-dimensional liquid chromatography tandem mass spectrometry. Paired t tests adjusted for multiple comparisons were used to assess differential expression between diacetyl and phosphate-buffered saline exposure. Of the significantly differentially expressed proteins identified, 61 were unique to the apical secretome, 81 were unique to the basolateral secretome, and 11 were present in both. Pathway enrichment analysis using publicly available databases revealed that proteins associated with matrix remodeling, including degradation, assembly, and new matrix organization, were overrepresented in the data sets. Similarly, protein modifiers of epidermal growth factor receptor signaling were significantly altered. The ordered changes in protein expression suggest that the airway epithelial response to diacetyl may contribute to BO pathogenesis.

Comparative inhalation toxicity of ethyltoluene isomers in rats and mice.

The C9 alkylbenzenes, composed mostly of ethyltoluenes and trimethylbenzenes, comprise 75-90% of the naphtha fraction of crude oil. Occupational and environmental exposure to C9 alkylbenzenes occur via inhalation. We conducted short-term inhalation studies on the ethyltoluene isomers (2-, 3- or 4-) to select one isomer for more comprehensive studies. Male Hsd:Sprague Dawley rats and female B6C3F1/N mice (n = 10) were exposed by nose-only inhalation to 2-, 3- or 4-ethyltoluene (0, 1000 or 2000 ppm) or cumene (a reference compound: 0, 500 or 1000 ppm) 3 h/day, 5 days/week, for 2 weeks. Clinical observations included abnormal gait and delayed righting reflex. Rats and mice exposed to 2000 ppm 2-ethyltoluene and mice exposed to 2000 ppm 4-ethyltoluene were euthanized early in moribund condition; no exposure-related deaths were observed with 3-ethyltoluene or cumene. Histopathology of selected tissues revealed that the nose and liver (rats and mice) and lung (mice only) to be toxicity targets. In the mouse lung, all compounds except 4-ethyltoluene produced bronchial and bronchiolar hyperplasia. In rats and mice, 2-ethyltoluene was the only compound to produce lesions in the nose and liver: in mice, squamous metaplasia and neutrophilic inflammation of the respiratory epithelium and atrophy and degeneration of the olfactory epithelium were observed in the nose and centrilobular hypertrophy and necrosis were observed in the liver. In rats, 2-ethyltoluene exposure produced atrophy of the olfactory epithelium in the nose and centrilobular necrosis in the liver. Based on mortality, body weight effects and histopathology, the 2-ethyltoluene isomer was the most potent isomer.

Comparative pulmonary toxicity of inhaled metalworking fluids in rats and mice.

Metalworking fluids (MWFs) are complex formulations designed for effective lubricating, cooling, and cleaning tools and parts during machining operations. Adverse health effects such as respiratory symptoms, dermatitis, and cancer have been reported in workers exposed to MWFs. Several constituents of MWFs have been implicated in toxicity and have been removed from the formulations over the years. However, animal studies with newer MWFs demonstrate that they continue to pose a health risk. This investigation examines the hypothesis that unrecognized health hazards exist in currently marketed MWF formulations that are presumed to be safe based on hazard assessments of individual ingredients. In vivo 13-week inhalation studies were designed to characterize and compare the potential toxicity of four MWFs: Trim VX, Cimstar 3800, Trim SC210, and Syntilo 1023. Male and female Wistar Han rats or Fischer 344N/Tac rats and B6C3F1/N mice were exposed to MWFs via whole-body inhalation at concentrations of 0, 25, 50, 100, 200, or 400 mg/m3 for 13 weeks, after which, survival, body and organ weights, hematology and clinical chemistry, histopathology, and genotoxicity were assessed following exposure. Although high concentrations were used, survival was not affected and toxicity was primarily within the respiratory tract of male and female rats and mice. Minor variances in toxicity were attributed to differences among species as well as in the chemical components of each MWF. Pulmonary fibrosis was present only in rats and mice exposed to Trim VX. These data confirm that newer MWFs have the potential to cause respiratory toxicity in workers who are repeatedly exposed via inhalation.

Proteomic Analysis of Primary Human Airway Epithelial Cells Exposed to the Respiratory Toxicant Diacetyl.

Occupational exposures to the diketone flavoring agent, diacetyl, have been associated with bronchiolitis obliterans, a rare condition of airway fibrosis. Model studies in rodents have suggested that the airway epithelium is a major site of diacetyl toxicity, but the effects of diacetyl exposure upon the human airway epithelium are poorly characterized. Here we performed quantitative LC-MS/MS-based proteomics to study the effects of repeated diacetyl vapor exposures on 3D organotypic cultures of human primary tracheobronchial epithelial cells. Using a label-free approach, we quantified approximately 3400 proteins and 5700 phosphopeptides in cell lysates across four independent donors. Altered expression of proteins and phosphopeptides were suggestive of loss of cilia and increased squamous differentiation in diacetyl-exposed cells. These phenomena were confirmed by immunofluorescence staining of culture cross sections. Hyperphosphorylation and cross-linking of basal cell keratins were also observed in diacetyl-treated cells, and we used parallel reaction monitoring to confidently localize and quantify previously uncharacterized sites of phosphorylation in keratin 6. Collectively, these data identify numerous molecular changes in the epithelium that may be important to the pathogenesis of flavoring-induced bronchiolitis obliterans. More generally, this study highlights the utility of quantitative proteomics for the study of in vitro models of airway injury and disease.

Pathology of diacetyl and 2,3-pentanedione airway lesions in a rat model of obliterative bronchiolitis.

Inhalation of diacetyl vapors by workers has been associated with obliterative bronchiolitis (OB), a poorly understood fibroproliferative disease of the small airways. Significant insights into the pathogenesis of OB have been obtained through the use of a rat model. Inhalation exposure of rats to diacetyl or 2,3-pentanedione, a related flavoring agent, can cause severe injury to the airway epithelium and underlying basement membrane. Repeated exposure to diacetyl or 2,3-pentanedione leads to aberrant repair, fibroproliferation and partial to complete occlusion of the airway lumen. Fibroproliferative lesions in rat airways were found to include both intraluminal polyps and circumferential intramural lesions. Intraluminal polyps have been observed to form secondary attachments spanning the airway lumen causing increasing obstruction. These airway lesions in rats are accompanied by inflammation in the form of peribronchial and perivascular infiltrates of lymphocytes, eosinophils and neutrophils. Diacetyl-induced OB lesions in the rat are similar to OB lesions in humans and provide a good model for studying the pathogenesis of this disease.

Hepatic transcriptomic alterations for N,N-dimethyl-p-toluidine (DMPT) and p-toluidine after 5-day exposure in rats.

N,N-dimethyl-p-toluidine (DMPT), an accelerant for methyl methacrylate monomers in medical devices, was a liver carcinogen in male and female F344/N rats and B6C3F1 mice in a 2-year oral exposure study. p-Toluidine, a structurally related chemical, was a liver carcinogen in mice but not in rats in an 18-month feed exposure study. In this current study, liver transcriptomic data were used to characterize mechanisms in DMPT and p-toluidine liver toxicity and for conducting benchmark dose (BMD) analysis. Male F344/N rats were exposed orally to DMPT or p-toluidine (0, 1, 6, 20, 60 or 120 mg/kg/day) for 5 days. The liver was examined for lesions and transcriptomic alterations. Both chemicals caused mild hepatic toxicity at 60 and 120 mg/kg and dose-related transcriptomic alterations in the liver. There were 511 liver transcripts differentially expressed for DMPT and 354 for p-toluidine at 120 mg/kg/day (false discovery rate threshold of 5 %). The liver transcriptomic alterations were characteristic of an anti-oxidative damage response (activation of the Nrf2 pathway) and hepatic toxicity. The top cellular processes in gene ontology (GO) categories altered in livers exposed to DMPT or p-toluidine were used for BMD calculations. The lower confidence bound benchmark doses for these chemicals were 2 mg/kg/day for DMPT and 7 mg/kg/day for p-toluidine. These studies show the promise of using 5-day target organ transcriptomic data to identify chemical-induced molecular changes that can serve as markers for preliminary toxicity risk assessment.

Molecular Changes in the Nasal Cavity after N, N-dimethyl-p-toluidine Exposure.

N, N-dimethyl-p-toluidine (DMPT; Cas No. 99-97-8), an accelerant for methyl methacrylate monomers in medical devices, is a nasal cavity carcinogen according to a 2-yr cancer study of male and female F344/N rats, with the nasal tumors arising from the transitional cell epithelium. In this study, we exposed male F344/N rats for 5 days to DMPT (0, 1, 6, 20, 60, or 120 mg/kg [oral gavage]) to explore the early changes in the nasal cavity after short-term exposure. Lesions occurred in the nasal cavity including hyperplasia of transitional cell epithelium (60 and 120 mg/kg). Nasal tissue was rapidly removed and preserved for subsequent laser capture microdissection and isolation of the transitional cell epithelium (0 and 120 mg/kg) for transcriptomic studies. DMPT transitional cell epithelium gene transcript patterns were characteristic of an antioxidative damage response (e.g., Akr7a3, Maff, and Mgst3), cell proliferation, and decrease in signals for apoptosis. The transcripts of amino acid transporters were upregulated (e.g., Slc7a11). The DMPT nasal transcript expression pattern was similar to that found in the rat nasal cavity after formaldehyde exposure, with over 1,000 transcripts in common. Molecular changes in the nasal cavity after DMPT exposure suggest that oxidative damage is a mechanism of the DMPT toxic and/or carcinogenic effects.

Chemical Reactivity and Respiratory Toxicity of the α-Diketone Flavoring Agents: 2,3-Butanedione, 2,3-Pentanedione, and 2,3-Hexanedione.

Occupational exposure to 2,3-butanedione (BD) vapors has been associated with severe respiratory disease leading to the use of potentially toxic substitutes. We compared the reactivity and respiratory toxicity of BD with that of two structurally related substitutes, 2,3-pentanedione (PD) and 2,3-hexanedione (HD). Chemical reactivity of the diketones with an arginine substrate decreased with increasing chain length (BD > PD > HD). Animals were evaluated the morning after a 2-week exposure to 0, 100, 150, or 200 ppm BD, PD, or HD (postexposure) or 2 weeks later (recovery). Bronchial fibrosis was observed in 5/5 BD and 5/5 PD rats at 200 ppm and in 4/6 BD and 6/6 PD rats at 150 ppm in the postexposure groups. Following recovery, bronchial fibrosis was observed in all surviving rats exposed to 200 ppm BD (5/5) or PD (3/3) and in 2/10 BD and 7/9 PD rats exposed to 150 ppm. Bronchial fibrosis was observed only in 2/12 HD-exposed rats in the 200 ppm postexposure group. Patchy interstitial fibrosis affected lungs of recovery groups exposed to 200 ppm PD (3/3) or BD (1/5) and to 150 ppm PD (4/9) or BD (7/10) and correlated with pulmonary function deficits. BD and PD were more reactive and produced more bronchial fibrosis than HD.

Impact of Environmental Enrichment Devices on NTP In Vivo Studies.

The goal of this study was to determine whether the use of nesting material or polycarbonate shelters as enrichment devices would have an impact on end points commonly measured during the conduct of the National Toxicology Program (NTP) 13-week studies. The study design was consistent with the NTP 13-week toxicity studies. Harlan Sprague-Dawley (HSD) rats and their offspring and B6C3F1/N mice were assigned to control (unenriched) and enriched experimental groups. Body weight, food and water consumption, behavioral observations, fecal content, clinical pathology, gross pathology, organ weights, and histopathology were evaluated. Enriched male mice and male and female rats exhibited decreased feed intake without a subsequent decrease in body weight; this may have been the result of the nesting material reducing the effect of cold stress, thereby allowing for more efficient use of feed. There were statistical differences in some hematological parameters; however, these were not considered physiologically relevant since all values were within the normal range. Gross pathology and histopathological findings were background changes and were not considered enrichment-related. Nesting material and shelters were used frequently and consistently and allowed animals to display species-typical behavior. There was no significant impact on commonly measured end points in HSD rats and B6C3F1/N mice given enrichment devices.

Gene expression in obliterative bronchiolitis-like lesions in 2,3-pentanedione-exposed rats.

Obliterative bronchiolitis (OB) is an irreversible lung disease characterized by progressive fibrosis in the small airways with eventual occlusion of the airway lumens. OB is most commonly associated with lung transplant rejection; however, OB has also been diagnosed in workers exposed to artificial butter flavoring (ABF) vapors. Research has been limited by the lack of an adequate animal model of OB, and as a result the mechanism(s) is unclear and there are no effective treatments for this condition. Exposure of rats to the ABF component, 2,3-pentanedione (PD) results in airway lesions that are histopathologically similar to those in human OB. We used this animal model to evaluate changes in gene expression in the distal bronchi of rats with PD-induced OB. Male Wistar Han rats were exposed to 200 ppm PD or air 6 h/d, 5 d/wk for 2-wks. Bronchial tissues were laser microdissected from serial sections of frozen lung. In exposed lungs, both fibrotic and non-fibrotic airways were collected. Following RNA extraction and microarray analysis, differential gene expression was evaluated. In non-fibrotic bronchi of exposed rats, 4683 genes were significantly altered relative to air-exposed controls with notable down-regulation of many inflammatory cytokines and chemokines. In contrast, in fibrotic bronchi, 3807 genes were significantly altered with a majority of genes being up-regulated in affected pathways. Tgf-ß2 and downstream genes implicated in fibrosis were significantly up-regulated in fibrotic lesions. Genes for collagens and extracellular matrix proteins were highly up-regulated. In addition, expression of genes for peptidases and peptidase inhibitors were significantly altered, indicative of the tissue remodeling that occurs during airway fibrosis. Our data provide new insights into the molecular mechanisms of OB. This new information is of potential significance with regard to future therapeutic targets for treatment.

Systemic uptake, albumin and hemoglobin binding of [(14)C]2,3-butanedione administered by intratracheal instillation in male Harlan Sprague Dawley rats and oropharyngeal aspiration in male B6C3F1/N mice.

2,3-Butanedione (BD) is a reactive diketone in artificial butter flavors that is thought to cause bronchiolitis obliterans in workers in microwave popcorn manufacturing. Bronchiolitis obliterans is generally not diagnosed until irreversible damage has occurred; therefore a biomarker of early exposure is needed. The potential systemic uptake of BD from inhalation exposure has not been evaluated. The objective here was to evaluate the systemic exposure of BD and binding to hemoglobin and albumin. [(14)C]BD was administered to male Harlan Sprague Dawley rats (100 mg/kg, intratracheal instillation) and B6C3F1/N mice (157 mg/kg, oropharyngeal aspiration). Blood and plasma was collected 24 h after administration and analyzed for (14)C content. At 24h, 0.88±0.07% of the administered dose was in rat blood, 0.66±0.06% in rat plasma, 0.38±0.13% in mouse blood and 0.17±0.05% in mouse plasma. Albumin binding in rats was 269±24.2 ng equiv./mg, which accounts for 38% of the radioactivity in plasma. In mice, binding was 85.0±22.3 ng equiv./mg albumin, which accounts for 51% of the radioactivity in plasma. The binding to hemoglobin in rats was 38.2±17.6 ng equiv./mg, and to globin was 29.1±3.96 ng equiv./mg. In mice, the binding to hemoglobin was 16.2±9.0 ng equiv./mg. The site(s) of adduction on hemoglobin and albumin was investigated by mass spectrometry. In rat globin, arginine adducts were detected at R-30 and R-104 of the beta chain in vitro and in vivo. In rat albumin, adducts were detected in vitro on R-219/221, R-360, and R-368, and in vivo on a variety of arginine residues. This study demonstrated that BD enters the systemic circulation and reacts with arginine on hemoglobin and albumin. These results indicate that hemoglobin and albumin adducts may be useful as biomarkers of BD exposure in humans.

Diversity Outbred Mice Identify Population-Based Exposure Thresholds and Genetic Factors that Influence Benzene-Induced Genotoxicity.

BACKGROUND: Inhalation of benzene at levels below the current exposure limit values leads to hematotoxicity in occupationally exposed workers. OBJECTIVE: We sought to evaluate Diversity Outbred (DO) mice as a tool for exposure threshold assessment and to identify genetic factors that influence benzene-induced genotoxicity. METHODS: We exposed male DO mice to benzene (0, 1, 10, or 100 ppm; 75 mice/exposure group) via inhalation for 28 days (6 hr/day for 5 days/week). The study was repeated using two independent cohorts of 300 animals each. We measured micronuclei frequency in reticulocytes from peripheral blood and bone marrow and applied benchmark concentration modeling to estimate exposure thresholds. We genotyped the mice and performed linkage analysis. RESULTS: We observed a dose-dependent increase in benzene-induced chromosomal damage and estimated a benchmark concentration limit of 0.205 ppm benzene using DO mice. This estimate is an order of magnitude below the value estimated using B6C3F1 mice. We identified a locus on Chr 10 (31.87 Mb) that contained a pair of overexpressed sulfotransferases that were inversely correlated with genotoxicity. CONCLUSIONS: The genetically diverse DO mice provided a reproducible response to benzene exposure. The DO mice display interindividual variation in toxicity response and, as such, may more accurately reflect the range of response that is observed in human populations. Studies using DO mice can localize genetic associations with high precision. The identification of sulfotransferases as candidate genes suggests that DO mice may provide additional insight into benzene-induced genotoxicity.

Macrophage solubilization and cytotoxicity of indium-containing particles as in vitro correlates to pulmonary toxicity in vivo.

Macrophage-solubilized indium-containing particles (ICPs) were previously shown in vitro to be cytotoxic. In this study, we compared macrophage solubilization and cytotoxicity of indium phosphide (InP) and indium-tin oxide (ITO) with similar particle diameters (∼ 1.5 µm) and then determined if relative differences in these in vitro parameters correlated with pulmonary toxicity in vivo. RAW 264.7 macrophages were treated with InP or ITO particles and cytotoxicity was assayed at 24 h. Ionic indium was measured in 24 h culture supernatants. Macrophage cytotoxicity and particle solubilization in vitro were much greater for InP compared with ITO. To correlate changes in vivo, B6C3F1 mice were treated with InP or ITO by oropharyngeal aspiration. On Days 14 and 28, bronchoalveolar lavage (BAL) and pleural lavage (PL) fluids were collected and assayed for total leukocytes. Cell differentials, lactate dehydrogenase activity, and protein levels were also measured in BAL. All lavage parameters were greatly increased in mice treated with InP compared with ITO. These data suggest that macrophage solubilization and cytotoxicity of some ICPs in vitro are capable of predicting pulmonary toxicity in vivo. In addition, these differences in toxicity were observed despite the two particulate compounds containing similar amounts of indium suggesting that solubilization, not total indium content, better reflects the toxic potential of some ICPs. Soluble InCl3 was shown to be more cytotoxic than InP to macrophages and lung epithelial cells in vitro further suggesting that ionic indium is the primary cytotoxic component of InP.

Diacetyl induces amphiregulin shedding in pulmonary epithelial cells and in experimental bronchiolitis obliterans.

Diacetyl (DA), a component of artificial butter flavoring, has been linked to the development of bronchiolitis obliterans (BO), a disease of airway epithelial injury and airway fibrosis. The epidermal growth factor receptor ligand, amphiregulin (AREG), has been implicated in other types of epithelial injury and lung fibrosis. We investigated the effects of DA directly on the pulmonary epithelium, and we hypothesized that DA exposure would result in epithelial cell shedding of AREG. Consistent with this hypothesis, we demonstrate that DA increases AREG by the pulmonary epithelial cell line NCI-H292 and by multiple independent primary human airway epithelial donors grown under physiologically relevant conditions at the air-liquid interface. Furthermore, we demonstrate that AREG shedding occurs through a TNF-α-converting enzyme (TACE)-dependent mechanism via inhibition of TACE activity in epithelial cells using the small molecule inhibitor, TNF-α protease inhibitor-1, as well as TACE-specific small inhibitor RNA. Finally, we demonstrate supportive in vivo results showing increased AREG transcript and protein levels in the lungs of rodents with DA-induced BO. In summary, our novel in vitro and in vivo observations suggest that further study of AREG is warranted in the pathogenesis of DA-induced BO.