Last data update: Apr 22, 2024. (Total: 46599 publications since 2009)
Records 1-30 (of 44 Records) |
Query Trace: Battelli L [original query] |
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Pulmonary evaluation of whole-body inhalation exposure of polycarbonate (PC) filament 3D printer emissions in rats
Farcas MT , McKinney W , Mandler WK , Knepp AK , Battelli L , Friend SA , Stefaniak AB , Service S , Kashon M , LeBouf RF , Thomas TA , Matheson J , Qian Y . J Toxicol Environ Health A 2024 87 (8) 325-341 During fused filament fabrication (FFF) 3D printing with polycarbonate (PC) filament, a release of ultrafine particles (UFPs) and volatile organic compounds (VOCs) occurs. This study aimed to determine PC filament printing emission-induced toxicity in rats via whole-body inhalation exposure. Male Sprague Dawley rats were exposed to a single concentration (0.529 mg/m(3), 40 nm mean diameter) of the 3D PC filament emissions in a time-course via whole body inhalation for 1, 4, 8, 15, and 30 days (4 hr/day, 4 days/week), and sacrificed 24 hr after the last exposure. Following exposures, rats were assessed for pulmonary and systemic responses. To determine pulmonary injury, total protein and lactate dehydrogenase (LDH) activity, surfactant proteins A and D, total as well as lavage fluid differential cells in bronchoalveolar lavage fluid (BALF) were examined, as well as histopathological analysis of lung and nasal passages was performed. To determine systemic injury, hematological differentials, and blood biomarkers of muscle, metabolic, renal, and hepatic functions were also measured. Results showed that inhalation exposure induced no marked pulmonary or systemic toxicity in rats. In conclusion, inhalation exposure of rats to a low concentration of PC filament emissions produced no significant pulmonary or systemic toxicity. |
Lung toxicity profile of inhaled copper-nickel welding fume in A/J mice
Zeidler-Erdely PC , Erdely A , Kodali V , Andrews R , Antonini J , Trainor-DeArmitt T , Salmen R , Battelli L , Grose L , Kashon M , Service S , McKinney W , Stone S , Falcone L . Inhal Toxicol 2022 34 1-12 Objective: Stainless steel welding creates fumes rich in carcinogenic metals such as chromium (Cr). Welding consumables devoid of Cr are being produced in an attempt to limit worker exposures to toxic and carcinogenic metals. The study objective was to characterize a copper-nickel (Cu-Ni) fume generated using gas metal arc welding (GMAW) and determine the pulmonary deposition and toxicity of the fume in mice exposed by inhalation. Materials and Methods: Male A/J mice (6-8 weeks of age) were exposed to air or Cu-Ni welding fumes for 2 (low deposition) or 4 (high deposition) hours/day for 10 days. Mice were sacrificed, and bronchoalveolar lavage (BAL), macrophage function, and histopathological analyses were performed at different timepoints post-exposure to evaluate resolution. Results and Discussion: Characterization of the fume indicated that most of the particles were between 0.1 and 1 µm in diameter, with a mass median aerodynamic diameter of 0.43 µm. Metal content of the fume was Cu (∼76%) and Ni (∼12%). Post-exposure, BAL macrophages had a reduced ability to phagocytose E. coli, and lung cytotoxicity was evident and significant (>12%-19% fold change). Loss of body weight was also significant at the early timepoints. Lung inflammation, the predominant finding identified by histopathology, was observed as a subacute response early that progressively resolved by 28 days with only macrophage aggregates remaining late (84 days). Conclusions: Overall, there was high acute lung toxicity with a resolution of the response in mice which suggests that the Cu-Ni fume may not be ideal for reducing toxic and inflammatory lung effects. |
Evaluation of pulmonary effects of 3-D printer emissions from acrylonitrile butadiene styrene using an air-liquid interface model of primary normal human-derived bronchial epithelial cells
Farcas MT , McKinney W , Coyle J , Orandle M , Mandler WK , Stefaniak AB , Bowers L , Battelli L , Richardson D , Hammer MA , Friend SA , Service S , Kashon M , Qi C , Hammond DR , Thomas TA , Matheson J , Qian Y . Int J Toxicol 2022 41 (4) 10915818221093605 This study investigated the inhalation toxicity of the emissions from 3-D printing with acrylonitrile butadiene styrene (ABS) filament using an air-liquid interface (ALI) in vitro model. Primary normal human-derived bronchial epithelial cells (NHBEs) were exposed to ABS filament emissions in an ALI for 4 hours. The mean and mode diameters of ABS emitted particles in the medium were 175 ± 24 and 153 ± 15 nm, respectively. The average particle deposition per surface area of the epithelium was 2.29 × 10(7) ± 1.47 × 10(7) particle/cm(2), equivalent to an estimated average particle mass of 0.144 ± 0.042 μg/cm(2). Results showed exposure of NHBEs to ABS emissions did not significantly affect epithelium integrity, ciliation, mucus production, nor induce cytotoxicity. At 24 hours after the exposure, significant increases in the pro-inflammatory markers IL-12p70, IL-13, IL-15, IFN-γ, TNF-α, IL-17A, VEGF, MCP-1, and MIP-1α were noted in the basolateral cell culture medium of ABS-exposed cells compared to non-exposed chamber control cells. Results obtained from this study correspond with those from our previous in vivo studies, indicating that the increase in inflammatory mediators occur without associated membrane damage. The combination of the exposure chamber and the ALI-based model is promising for assessing 3-D printer emission-induced toxicity. |
Histopathology of the broad class of carbon nanotubes and nanofibers used or produced in U.S. facilities in a murine model
Fraser K , Hubbs A , Yanamala N , Mercer RR , Stueckle TA , Jensen J , Eye T , Battelli L , Clingerman S , Fluharty K , Dodd T , Casuccio G , Bunker K , Lersch TL , Kashon ML , Orandle M , Dahm M , Schubauer-Berigan MK , Kodali V , Erdely A . Part Fibre Toxicol 2021 18 (1) 47 BACKGROUND: Multi-walled carbon nanotubes and nanofibers (CNT/F) have been previously investigated for their potential toxicities; however, comparative studies of the broad material class are lacking, especially those with a larger diameter. Additionally, computational modeling correlating physicochemical characteristics and toxicity outcomes have been infrequently employed, and it is unclear if all CNT/F confer similar toxicity, including histopathology changes such as pulmonary fibrosis. Male C57BL/6 mice were exposed to 40 µg of one of nine CNT/F (MW #1-7 and CNF #1-2) commonly found in exposure assessment studies of U.S. facilities with diameters ranging from 6 to 150 nm. Human fibroblasts (0-20 µg/ml) were used to assess the predictive value of in vitro to in vivo modeling systems. RESULTS: All materials induced histopathology changes, although the types and magnitude of the changes varied. In general, the larger diameter MWs (MW #5-7, including Mitsui-7) and CNF #1 induced greater histopathology changes compared to MW #1 and #3 while MW #4 and CNF #2 were intermediate in effect. Differences in individual alveolar or bronchiolar outcomes and severity correlated with physical dimensions and how the materials agglomerated. Human fibroblast monocultures were found to be insufficient to fully replicate in vivo fibrosis outcomes suggesting in vitro predictive potential depends upon more advanced cell culture in vitro models. Pleural penetrations were observed more consistently in CNT/F with larger lengths and diameters. CONCLUSION: Physicochemical characteristics, notably nominal CNT/F dimension and agglomerate size, predicted histopathologic changes and enabled grouping of materials by their toxicity profiles. Particles of greater nominal tube length were generally associated with increased severity of histopathology outcomes. Larger particle lengths and agglomerates were associated with more severe bronchi/bronchiolar outcomes. Spherical agglomerated particles of smaller nominal tube dimension were linked to granulomatous inflammation while a mixture of smaller and larger dimensional CNT/F resulted in more severe alveolar injury. |
Pulmonary and systemic toxicity in rats following inhalation exposure of 3-D printer emissions from acrylonitrile butadiene styrene (ABS) filament
Farcas MT , McKinney W , Qi C , Mandler KW , Battelli L , Friend SA , Stefaniak AB , Jackson M , Orandle M , Winn A , Kashon M , LeBouf RF , Russ KA , Hammond DR , Burns D , Ranpara A , Thomas TA , Matheson J , Qian Y . Inhal Toxicol 2020 32 1-16 BACKGROUND: Fused filament fabrication 3-D printing with acrylonitrile butadiene styrene (ABS) filament emits ultrafine particulates (UFPs) and volatile organic compounds (VOCs). However, the toxicological implications of the emissions generated during 3-D printing have not been fully elucidated. AIM AND METHODS: The goal of this study was to investigate the in vivo toxicity of ABS-emissions from a commercial desktop 3-D printer. Male Sprague Dawley rats were exposed to a single concentration of ABS-emissions or air for 4 hours/day, 4 days/week for five exposure durations (1, 4, 8, 15, and 30 days). At 24 hours after the last exposure, rats were assessed for pulmonary injury, inflammation, and oxidative stress as well as systemic toxicity. RESULTS AND DISCUSSION: 3-D printing generated particulate with average particle mass concentration of 240 ± 90 µg/m³, with an average geometric mean particle mobility diameter of 85 nm (geometric standard deviation = 1.6). The number of macrophages increased significantly at day 15. In bronchoalveolar lavage, IFN-γ and IL-10 were significantly higher at days 1 and 4, with IL-10 levels reaching a peak at day 15 in ABS-exposed rats. Neither pulmonary oxidative stress responses nor histopathological changes of the lungs and nasal passages were found among the treatments. There was an increase in platelets and monocytes in the circulation at day 15. Several serum biomarkers of hepatic and kidney functions were significantly higher at day 1. CONCLUSIONS: At the current experimental conditions applied, it was concluded that the emissions from ABS filament caused minimal transient pulmonary and systemic toxicity. |
Tumorigenic response in lung tumor susceptible A/J mice after sub-chronic exposure to calcium chromate or iron (III) oxide
Zeidler-Erdely PC , Falcone LM , Antonini JM , Fraser K , Kashon ML , Battelli LA , Salmen R , Trainor T , Grose L , Friend S , Yang C , Erdely A . Toxicol Lett 2020 334 60-65 Iron oxides are Group 3 (not classifiable as to its carcinogenicity to humans) according to the International Agency for Research on Cancer (IARC). Occupational exposures during iron and steel founding and hematite underground mining as well as other iron predominant exposures such as welding are Group 1 (carcinogenic to humans). The objective of this study was to investigate the potential of iron as iron (III) oxide (Fe(2)O(3)) to initiate lung tumors in A/J mice, a lung tumor susceptible strain. Male A/J mice were exposed by oropharyngeal aspiration to suspensions of Fe(2)O(3) (1 mg) or calcium chromate (CaCrO(4); 100 µg; positive control) for 26 weeks (once per week). Shams were exposed to 50 µL phosphate buffered saline (PBS; vehicle). Mice were euthanized 70 weeks after the first exposure and lung nodules were enumerated. Both CaCrO(4) and Fe(2)O(3) significantly increased gross-observed lung tumor multiplicity in A/J mice (9.63 ± 0.55 and 3.35 ± 0.30, respectively) compared to sham (2.31 ± 0.19). Histopathological analysis showed that bronchiolo-alveolar adenomas (BAA) and carcinomas (BAC) were the primary lung tumor types in all groups and were increased in the exposed groups compared to sham. BAC were significantly increased (146 %) in the CaCrO(4) group and neared significance in the Fe(2)O(3) group (100 % increase; p = 0.085). BAA and other histopathological indices of toxicity followed the same pattern with exposed groups increased compared to sham control. In conclusion, evidence from this study, in combination with our previous studies, demonstrate that exposure to iron alone may be a potential risk factor for lung carcinogenesis. |
Resolution of pulmonary inflammation induced by carbon nanotubes and fullerenes in mice: Role of macrophage polarization
Lim CS , Porter DW , Orandle MS , Green BJ , Barnes MA , Croston TL , Wolfarth MG , Battelli LA , Andrew ME , Beezhold DH , Siegel PD , Ma Q . Front Immunol 2020 11 1186 Pulmonary exposure to certain engineered nanomaterials (ENMs) causes chronic lesions like fibrosis and cancer in animal models as a result of unresolved inflammation. Resolution of inflammation involves the time-dependent biosynthesis of lipid mediators (LMs)-in particular, specialized pro-resolving mediators (SPMs). To understand how ENM-induced pulmonary inflammation is resolved, we analyzed the inflammatory and pro-resolving responses to fibrogenic multi-walled carbon nanotubes (MWCNTs, Mitsui-7) and low-toxicity fullerenes (fullerene C60, C60F). Pharyngeal aspiration of MWCNTs at 40 mug/mouse or C60F at a dose above 640 mug/mouse elicited pulmonary effects in B6C3F1 mice. Both ENMs stimulated acute inflammation, predominated by neutrophils, in the lung at day 1, which transitioned to histiocytic inflammation by day 7. By day 28, the lesion in MWCNT-exposed mice progressed to fibrotic granulomas, whereas it remained as alveolar histiocytosis in C60F-exposed mice. Flow cytometric profiling of whole lung lavage (WLL) cells revealed that neutrophil recruitment was the greatest at day 1 and declined to 36.6% of that level in MWCNT- and 16.8% in C60F-treated mice by day 7, and to basal levels by day 28, suggesting a rapid initiation phase and an extended resolution phase. Both ENMs induced high levels of proinflammatory leukotriene B4 (LTB4) and prostaglandin E2 (PGE2) with peaks at day 1, and high levels of SPMs resolvin D1 (RvD1) and E1 (RvE1) with peaks at day 7. MWCNTs and C60F induced time-dependent polarization of M1 macrophages with a peak at day 1 and subsequently of M2 macrophages with a peak at day 7 in the lung, accompanied by elevated levels of type 1 or type 2 cytokines, respectively. M1 macrophages exhibited preferential induction of arachidonate 5-lipoxygenase activating protein (ALOX5AP), whereas M2 macrophages had a high level expression of arachidonate 15-lipoxygenase (ALOX15). Polarization of macrophages in vitro differentially induced ALOX5AP in M1 macrophages or ALOX15 in M2 macrophages resulting in increased preferential biosynthesis of proinflammatory LMs or SPMs. MWCNTs increased the M1- or M2-specific production of LMs accordingly. These findings support a mechanism by which persistent ENM-induced neutrophilic inflammation is actively resolved through time-dependent polarization of macrophages and enhanced biosynthesis of specialized LMs via distinct ALOX pathways. |
Mouse pulmonary dose- and time course-responses induced by exposure to nitrogen-doped multi-walled carbon nanotubes
Porter DW , Orandle M , Zheng P , Wu N , Hamilton RF Jr , Holian A , Chen BT , Andrew M , Wolfarth MG , Battelli L , Tsuruoka S , Terrones M , Castranova V . Inhal Toxicol 2020 32 (1) 1-15 Objective: In this study, we compared in vitro and in vivo bioactivity of nitrogen-doped multi-walled carbon nanotubes (NDMWCNT) to MWCNT to test the hypothesis that nitrogen doping would alter bioactivity.Materials and Methods: High-resolution transmission electron microscopy (TEM) confirmed the multilayer structure of MWCNT with an average layer distance of 0.36 nm, which was not altered by nitrogen doping: the nanomaterials had similar widths and lengths. In vitro studies with THP-1 cells and alveolar macrophages from C57BL/6 mice demonstrated that NDMWCNT were less cytotoxic and stimulated less IL-1beta release compared to MWCNT. For in vivo studies, male C57BL/6J mice received a single dose of dispersion medium (DM), 2.5, 10 or 40 microg/mouse of NDMWCNT, or 40 microg/mouse of MWCNT by oropharyngeal aspiration. Animals were euthanized between 1 and 7 days post-exposure for whole lung lavage (WLL) studies.Results and Discussion: NDMWCNT caused time- and dose-dependent pulmonary inflammation. However, it was less than that caused by MWCNT. Activation of the NLRP3 inflammasome was assessed in particle-exposed mice by determining cytokine production in WLL fluid at 1 day post-exposure. Compared to DM-exposed mice, IL-1beta and IL-18 were significantly increased in MWCNT- and NDMWCNT-exposed mice, but the increase caused by NDMWCNT was less than MWCNT. At 56 days post-exposure, histopathology determined lung fibrosis in MWCNT-exposed mice was greater than NDMWCNT-exposed mice.Conclusions: These data indicate nitrogen doping of MWCNT decreases their bioactivity, as reflected with lower in vitro and in vivo toxicity inflammation and lung disease. The lower activation of the NLRP3 inflammasome may be responsible. Abbreviations: NDMWCNT: nitrogen-doped multi-walled carbon nanotubes; MWCNT: multi-walled carbon nanotubes; TEM: transmission electron microscopy; HRTEM: high resolution transmission electron microscopy; IL-1ss: interleukin-1ss; DM: dispersion medium; WLL: whole lung lavage; IL-18: interleukin-18; GSD: geometric standard deviation; XPS: X-ray photoelectron spectroscopy; SEM: standard error of the mean; PMA: phorbol 12-myristate 13-acetate; LPS: lipopolysacharride; LDH: lactate dehydrogenase; AM: alveolar macrophage; PMN: polymorphonuclear leukocyte. |
Flavorings-related lung disease: A brief review and new mechanistic data
Hubbs AF , Kreiss K , Cummings KJ , Fluharty KL , O'Connell R , Cole A , Dodd TM , Clingerman SM , Flesher JR , Lee R , Pagel S , Battelli LA , Cumpston A , Jackson M , Kashon M , Orandle MS , Fedan JS , Sriram K . Toxicol Pathol 2019 47 (8) 192623319879906 Flavorings-related lung disease is a potentially disabling and sometimes fatal lung disease of workers making or using flavorings. First identified almost 20 years ago in microwave popcorn workers exposed to butter-flavoring vapors, flavorings-related lung disease remains a concern today. In some cases, workers develop bronchiolitis obliterans, a severe form of fixed airways disease. Affected workers have been reported in microwave popcorn, flavorings, and coffee production workplaces. Volatile alpha-dicarbonyl compounds, particularly diacetyl (2,3-butanedione) and 2,3-pentanedione, are implicated in the etiology. Published studies on diacetyl and 2,3-pentanedione document their ability to cause airway epithelial necrosis, damage biological molecules, and perturb protein homeostasis. With chronic exposure in rats, they produce airway fibrosis resembling bronchiolitis obliterans. To add to this knowledge, we recently evaluated airway toxicity of the 3-carbon alpha-dicarbonyl compound, methylglyoxal. Methylglyoxal inhalation causes epithelial necrosis at even lower concentrations than diacetyl. In addition, we investigated airway toxicity of mixtures of diacetyl, acetoin, and acetic acid, common volatiles in butter flavoring. At ratios comparable to workplace scenarios, the mixtures or diacetyl alone, but not acetic acid or acetoin, cause airway epithelial necrosis. These new findings add to existing data to implicate alpha-dicarbonyl compounds in airway injury and flavorings-related lung disease. |
Mouse pulmonary response to dust from sawing Corian(R), a solid-surface composite material
Mandler WK , Qi C , Orandle MS , Sarkisian K , Mercer RR , Stefaniak AB , Knepp AK , Bowers LN , Battelli LA , Shaffer J , Friend SA , Qian Y , Sisler JD . J Toxicol Environ Health A 2019 82 (11) 1-19 Corian(R), a solid-surface composite (SSC), is composed of alumina trihydrate and acrylic polymer. The aim of the present study was to examine the pulmonary toxicity attributed to exposure to SSC sawing dust. Male mice were exposed to either phosphate buffer saline (PBS, control), 62.5, 125, 250, 500, or 1000 microg of SSC dust, or 1000 microg silica (positive control) via oropharyngeal aspiration. Body weights were measured for the duration of the study. Bronchoalveolar lavage fluid (BALF) and tissues were collected for analysis at 1 and 14 days post-exposure. Enhanced-darkfield and histopathologic analysis was performed to assess particle distribution and inflammatory responses. BALF cells and inflammatory cytokines were measured. The geometric mean diameter of SSC sawing dust following suspension in PBS was 1.25 microm. BALF analysis indicated that lactate dehydrogenase (LDH) activity, inflammatory cells, and pro-inflammatory cytokines were significantly elevated in the 500 and 1000 microg SSC exposure groups at days 1 and 14, suggesting that exposure to these concentrations of SSC induced inflammatory responses, in some cases to a greater degree than the silica positive control. Histopathology indicated the presence of acute alveolitis at all doses at day 1, which was largely resolved by day 14. Alveolar particle deposition and granulomatous mass formation were observed in all exposure groups at day 14. The SSC particles were poorly cleared, with 81% remaining at the end of the observation period. These findings demonstrate that SSC sawing dust exposure induces pulmonary inflammation and damage that warrants further investigation. Abbreviations: ANOVA: Analysis of Variance; ATH: Alumina Trihydrate; BALF: Bronchoalveolar Lavage Fluid; Dpg: Geometric Mean Diameter; FE-SEM: Field Emission Scanning Electron Microscopy; IACUC: Institutional Animal Care and Use Committee; IFN-gamma: Interferon Gamma; IL-1 Beta: Interleukin-1 Beta; IL-10: Interleukin-10; IL-12: Interleukin-12; IL-2: Interleukin-2; IL-4: Interleukin-4; IL-5: Interleukin-5; IL-6: Interleukin-6; KC/GRO: Neutrophil-Activating Protein 3; MMAD: Mass Median Aerodynamic Diameter; PBS: Phosphate-Buffered Saline; PEL: Permissible Exposure Limit; PM: Polymorphonuclear Leukocytes; PNOR: Particles Not Otherwise Regulated; SEM/EDX: Scanning Electron Microscope/Energy-Dispersive X-Ray; SSA: Specific Surface Area; SSC: Solid Surface Composite; TNFalpha: Tumor Necrosis Factor-Alpha; VOC: Volatile Organic Compounds; sigmag: Geometric Standard Deviation. |
Toxicological assessment of dust from sanding micronized copper-treated lumber in vivo
Sisler JD , Mandler WK , Shaffer J , Lee T , McKinney WG , Battelli LA , Orandle MS , Thomas TA , Castranova VC , Qi C , Porter DW , Andrew ME , Fedan JS , Mercer RR , Qian Y . J Hazard Mater 2019 373 630-639 Micronized copper azole (MCA) is a lumber treatment improve longevity. In this study, the in vivo response to PM2.5 sanding dust generated from MCA-treated lumber was compared to that of untreated yellow pine (UYP) or soluble copper azole-treated (CA-C) lumber to determine if the MCA was more bioactive than CA-C. Mice were exposed to doses (28, 140, or 280 mug/mouse) of UYP, MCA, or CA-C sanding dust using oropharyngeal aspiration. Bronchoalveolar lavage fluid (BALF) lactate dehydrogenase activity was increased at 1 day post-exposure to 280 mug/mouse of MCA and CA-C compared to UYP. BALF polymorphonuclear cells were increased by MCA and CA-C. There were increases in BALF cytokines in MCA and CA-C-exposed groups at 1 day post-exposure. Lung histopathology indicated inflammation with infiltration of neutrophils and macrophages. Pulmonary responses were more severe in MCA and CA-C-exposed groups at 1 day post-exposure. MCA caused more severe inflammatory responses than CA-C at 1 day post-exposure. These findings suggest that the MCA and CA-C sanding dusts are more bioactive than the UYP sanding dust, and, moreover, the MCA sanding dust is more bioactive in comparison to the CA-C sanding dust. No chronic toxic effects were observed among all observed sanding dusts. |
Pulmonary toxicity and lung tumorigenic potential of surrogate metal oxides in gas metal arc welding-stainless steel fume: Iron as a primary mediator versus chromium and nickel
Falcone LM , Erdely A , Salmen R , Keane M , Battelli L , Kodali V , Bowers L , Stefaniak AB , Kashon ML , Antonini JM , Zeidler-Erdely PC . PLoS One 2018 13 (12) e0209413 In 2017, the International Agency for Research on Cancer classified welding fumes as "carcinogenic to humans" (Group 1). Both mild steel (MS) welding, where fumes lack carcinogenic chromium and nickel, and stainless steel (SS) increase lung cancer risk in welders; therefore, further research to better understand the toxicity of the individual metals is needed. The objectives were to (1) compare the pulmonary toxicity of chromium (as Cr(III) oxide [Cr2O3] and Cr (VI) calcium chromate [CaCrO4]), nickel [II] oxide (NiO), iron [III] oxide (Fe2O3), and gas metal arc welding-SS (GMAW-SS) fume; and (2) determine if these metal oxides can promote lung tumors. Lung tumor susceptible A/J mice (male, 4-5 weeks old) were exposed by oropharyngeal aspiration to vehicle, GMAW-SS fume (1.7 mg), or a low or high dose of surrogate metal oxides based on the respective weight percent of each metal in the fume: Cr2O3 + CaCrO4 (366 + 5 mug and 731 + 11 mug), NiO (141 and 281 mug), or Fe2O3 (1 and 2 mg). Bronchoalveolar lavage, histopathology, and lung/liver qPCR were done at 1, 7, 28, and 84 days post-aspiration. In a two-stage lung carcinogenesis model, mice were initiated with 3-methylcholanthrene (10 mug/g; intraperitoneal; 1x) or corn oil then exposed to metal oxides or vehicle (1 x/week for 5 weeks) by oropharyngeal aspiration. Lung tumors were counted at 30 weeks post-initiation. Results indicate the inflammatory potential of the metal oxides was Fe2O3 > Cr2O3 + CaCrO4 > NiO. Overall, the pneumotoxic effects were negligible for NiO, acute but not persistent for Cr2O3 + CaCrO4, and persistent for the Fe2O3 exposures. Fe2O3, but not Cr2O3 + CaCrO4 or NiO significantly promoted lung tumors. These results provide experimental evidence that Fe2O3 is an important mediator of welding fume toxicity and support epidemiological findings and the IARC classification. |
Inhalation of iron-abundant gas metal arc welding-mild steel fume promotes lung tumors in mice
Falcone LM , Erdely A , Kodali V , Salmen R , Battelli LA , Dodd T , McKinney W , Stone S , Donlin M , Leonard HD , Cumpston JL , Cumpston JB , Andrews RN , Kashon ML , Antonini JM , Zeidler-Erdely PC . Toxicology 2018 409 24-32 Welding fumes were reclassified as a Group 1 carcinogen by the International Agency for Research on Cancer in 2017. Gas metal arc welding (GMAW) is a process widely used in industry. Fume generated from GMAW-mild steel (MS) is abundant in iron with some manganese, while GMAW-stainless steel (SS) fume also contains significant amounts of chromium and nickel, known carcinogenic metals. It has been shown that exposure to GMAW-SS fume in A/J mice promotes lung tumors. The objective was to determine if GMAW-MS fume, which lacks known carcinogenic metals, also promotes lung tumors in mice. Male A/J mice received a single intraperitoneal injection of corn oil or the initiator 3-methylcholanthrene (MCA; 10 mug/g) and, one week later, were exposed by whole-body inhalation to GMAW-MS aerosols for 4 hours/day x 4 days/week x 8 weeks at a mean concentration of 34.5 mg/m(3). Lung nodules were enumerated by gross examination at 30 weeks post-initiation. GMAW-MS fume significantly increased lung tumor multiplicity in mice initiated with MCA (21.86 +/- 1.50) compared to MCA/air-exposed mice (8.34 +/- 0.59). Histopathological analysis confirmed these findings and also revealed an absence of inflammation. Bronchoalveolar lavage analysis also indicated a lack of lung inflammation and toxicity after short-term inhalation exposure to GMAW-MS fume. In conclusion, this study demonstrates that inhalation of GMAW-MS fume promotes lung tumors in vivo and aligns with epidemiologic evidence that shows MS welders, despite less exposure to carcinogenic metals, are at an increased risk for lung cancer. |
Short-term pulmonary toxicity assessment of pre- and post-incinerated organomodified nanoclay in mice
Stueckle TA , Davidson DC , Derk R , Kornberg TG , Battelli L , Friend S , Orandle M , Wagner A , Dinu CZ , Sierros KA , Agarwal S , Gupta RK , Rojanasakul Y , Porter DW , Rojanasakul L . ACS Nano 2018 12 (3) 2292-2310 Organomodified nanoclays (ONCs) are increasingly used as filler materials to improve nanocomposite strength, wettability, flammability, and durability. However, pulmonary risks associated with exposure along their chemical lifecycle are unknown. This study's objective was to compare pre- and post-incinerated forms of uncoated and organomodified nanoclays for potential pulmonary inflammation, toxicity, and systemic blood response. Mice were exposed via aspiration to low (30 mug) and high (300 mug) doses of preincinerated uncoated montmorillonite nanoclay (CloisNa), ONC (Clois30B), their respective incinerated forms (I-CloisNa and I-Clois30B), and crystalline silica (CS). Lung and blood tissues were collected at days 1, 7, and 28 to compare toxicity and inflammation indices. Well-dispersed CloisNa caused a robust inflammatory response characterized by neutrophils, macrophages, and particle-laden granulomas. Alternatively, Clois30B, I-Clois30B, and CS high-dose exposures elicited a low grade, persistent inflammatory response. High-dose Clois30B exposure exhibited moderate increases in lung damage markers and a delayed macrophage recruitment cytokine signature peaking at day 7 followed by a fibrotic tissue signature at day 28, similar to CloisNa. I-CloisNa exhibited acute, transient inflammation with quick recovery. Conversely, high-dose I-Clois30B caused a weak initial inflammatory signal but showed comparable pro-inflammatory signaling to CS at day 28. The data demonstrate that ONC pulmonary toxicity and inflammatory potential relies on coating presence and incineration status in that coated and incinerated nanoclay exhibited less inflammation and granuloma formation than pristine montmorillonite. High doses of both pre- and post-incinerated ONC, with different surface morphologies, may harbor potential pulmonary health hazards over long-term occupational exposures. |
The fate of inhaled nanoparticles: Detection and measurement by enhanced dark-field microscopy
Mercer RR , Scabilloni JF , Wang L , Battelli LA , Antonini JM , Roberts JR , Qian Y , Sisler JD , Castranova V , Porter DW , Hubbs AF . Toxicol Pathol 2017 46 (1) 192623317732321 Assessing the potential health risks for newly developed nanoparticles poses a significant challenge. Nanometer-sized particles are not generally detectable with the light microscope. Electron microscopy typically requires high-level doses, above the physiologic range, for particle examination in tissues. Enhanced dark-field microscopy (EDM) is an adaption of the light microscope that images scattered light. Nanoparticles scatter light with high efficiency while normal tissues do not. EDM has the potential to identify the critical target sites for nanoparticle deposition and injury in the lungs and other organs. This study describes the methods for EDM imaging of nanoparticles and applications. Examples of EDM application include measurement of deposition and clearance patterns. Imaging of a wide variety of nanoparticles demonstrated frequent situations where nanoparticles detected by EDM were not visible by light microscopy. EDM examination of colloidal gold nanospheres (10-100 nm diameter) demonstrated a detection size limit of approximately 15 nm in tissue sections. EDM determined nanoparticle volume density was directly proportional to total lung burden of exposed animals. The results confirm that EDM can determine nanoparticle distribution, clearance, transport to lymph nodes, and accumulation in extrapulmonary organs. Thus, EDM substantially improves the qualitative and quantitative microscopic evaluation of inhaled nanoparticles. |
In Vivo Toxicity Assessment of Occupational Components of the Carbon Nanotube Life Cycle To Provide Context to Potential Health Effects
Bishop L , Cena L , Orandle M , Yanamala N , Dahm MM , Birch ME , Evans DE , Kodali VK , Eye T , Battelli L , Zeidler-Erdely PC , Casuccio G , Bunker K , Lupoi JS , Lersch TL , Stefaniak AB , Sager T , Afshari A , Schwegler-Berry D , Friend S , Kang J , Siegrist KJ , Mitchell CA , Lowry DT , Kashon ML , Mercer RR , Geraci CL , Schubauer-Berigan MK , Sargent LM , Erdely A . ACS Nano 2017 11 (9) 8849-8863 Pulmonary toxicity studies on carbon nanotubes focus primarily on as-produced materials and rarely are guided by a life cycle perspective or integration with exposure assessment. Understanding toxicity beyond the as-produced, or pure native material, is critical, due to modifications needed to overcome barriers to commercialization of applications. In the first series of studies, the toxicity of as-produced carbon nanotubes and their polymer-coated counterparts was evaluated in reference to exposure assessment, material characterization, and stability of the polymer coating in biological fluids. The second series of studies examined the toxicity of aerosols generated from sanding polymer-coated carbon-nanotube-embedded or neat composites. Postproduction modification by polymer coating did not enhance pulmonary injury, inflammation, and pathology or in vitro genotoxicity of as-produced carbon nanotubes, and for a particular coating, toxicity was significantly attenuated. The aerosols generated from sanding composites embedded with polymer-coated carbon nanotubes contained no evidence of free nanotubes. The percent weight incorporation of polymer-coated carbon nanotubes, 0.15% or 3% by mass, and composite matrix utilized altered the particle size distribution and, in certain circumstances, influenced acute in vivo toxicity. Our study provides perspective that, while the number of workers and consumers increases along the life cycle, toxicity and/or potential for exposure to the as-produced material may greatly diminish. |
Lung bioactivity of vapor grown carbon nanofibers
Porter DW , Orandle M , Mercer RR , Wu N , Zheng P , Chen BT , Holian A , Andrew M , Leonard S , Wolfarth M , Friend S , Battelli L , Hamilton RF Jr , Hagiwara Y , Koyama T , Castranova V . NanoImpact 2017 6 1-10 Vapor grown carbon nanofibers (VGCF-H) is an example of a two dimensional carbon based nanoparticle. In the present study, male C57Bl/6J mice were exposed to VGCF-H (10–80 μg) by pharyngeal aspiration; dispersion medium (DM) was used as the vehicle. At 1, 7 and 28 days post-exposure, lung lavage and histopathology studies were conducted. VGCF-H cytotoxicity was assessed by measuring acellular lavage fluid lactate dehydrogenase (LDH) activity, and determined that VGCF-H exposure produced dose-dependent increases in LDH activity which decreased over time. Using polymorphonuclear leukocytes as a marker, VGCF-H-exposure produced dose-dependent lung inflammation which decreased over time. Histologically, the incidence and severity of pulmonary inflammation was confirmed to be dose-dependent, and inflammatory infiltrates were characterized by increased numbers of alveolar macrophages with small numbers of neutrophils. VGCF-H caused dose- and time-dependent increases in cathepsin activity and cytokines in the acellular lavage fluid, indicating activation of the NLRP3 inflammasome by VGCFH may contribute to lung inflammation. VGCF-H exposure caused minimal to mild interstitial alveolar fibrosis, characterized by increased amounts of collagen fibers in the interstitium, and the incidence and severity of fibrosis tended to increase with the VGCF-H dose. Accumulation of VGCF-H fibers in the tracheobronchial lymph nodes was observed by 28 days after exposure at 40 and 80 μg doses. |
Inhalation of gas metal arc-stainless steel welding fume promotes lung tumorigenesis in A/J mice
Falcone LM , Erdely A , Meighan TG , Battelli LA , Salmen R , McKinney W , Stone S , Cumpston A , Cumpston J , Andrews RN , Kashon M , Antonini JM , Zeidler-Erdely PC . Arch Toxicol 2017 91 (8) 2953-2962 Epidemiologic studies suggest an increased risk of lung cancer with exposure to welding fumes, but controlled animal studies are needed to support this association. Oropharyngeal aspiration of collected "aged" gas metal arc-stainless steel (GMA-SS) welding fume has been shown by our laboratory to promote lung tumor formation in vivo using a two-stage initiation-promotion model. Our objective in this study was to determine whether inhalation of freshly generated GMA-SS welding fume also acts as a lung tumor promoter in lung tumor-susceptible mice. Male A/J mice received intraperitoneal (IP) injections of corn oil or the chemical initiator 3-methylcholanthrene (MCA; 10 microg/g) and 1 week later were exposed by whole-body inhalation to air or GMA-SS welding aerosols for 4 h/d x 4 d/w x 9 w at a target concentration of 40 mg/m3. Lung nodules were enumerated at 30 weeks post-initiation. GMA-SS fume significantly promoted lung tumor multiplicity in A/J mice initiated with MCA (16.11 +/- 1.18) compared to MCA/air-exposed mice (7.93 +/- 0.82). Histopathological analysis found that the increased number of lung nodules in the MCA/GMA-SS group were hyperplasias and adenomas, which was consistent with developing lung tumorigenesis. Metal deposition analysis in the lung revealed a lower deposited dose, approximately fivefold compared to our previous aspiration study, still elicited a significant lung tumorigenic response. In conclusion, this study demonstrates that inhaling GMA-SS welding fume promotes lung tumorigenesis in vivo which is consistent with the epidemiologic studies that show welders may be at an increased risk for lung cancer. |
Accumulation of ubiquitin and sequestosome-1 implicate protein damage in diacetyl-induced cytotoxicity
Hubbs AF , Fluharty KL , Edwards RJ , Barnabei JL , Grantham JT , Palmer SM , Kelly F , Sargent LM , Reynolds SH , Mercer RR , Goravanahally MP , Kashon ML , Honaker JC , Jackson MC , Cumpston AM , Goldsmith WT , McKinney W , Fedan JS , Battelli LA , Munro T , Bucklew-Moyers W , McKinstry K , Schwegler-Berry D , Friend S , Knepp AK , Smith SL , Sriram K . Am J Pathol 2016 186 (11) 2887-2908 Inhaled diacetyl vapors are associated with flavorings-related lung disease, a potentially fatal airway disease. The reactive alpha-dicarbonyl group in diacetyl causes protein damage in vitro. Dicarbonyl/l-xylulose reductase (DCXR) metabolizes diacetyl into acetoin, which lacks this alpha-dicarbonyl group. To investigate the hypothesis that flavorings-related lung disease is caused by in vivo protein damage, we correlated diacetyl-induced airway damage in mice with immunofluorescence for markers of protein turnover and autophagy. Western immunoblots identified shifts in ubiquitin pools. Diacetyl inhalation caused dose-dependent increases in bronchial epithelial cells with puncta of both total ubiquitin and K63-ubiquitin, central mediators of protein turnover. This response was greater in Dcxr-knockout mice than in wild-type controls inhaling 200 ppm diacetyl, further implicating the alpha-dicarbonyl group in the protein damage. Western immunoblots demonstrated decreased free ubiquitin in airway-enriched fractions. Transmission electron microscopy and colocalization of ubiquitin-positive puncta with lysosomal markers lysosomal-associated membrane protein 1 and 2 and with the multifunctional scaffolding protein sequestosome-1 (SQSTM1/p62) confirmed autophagy. Surprisingly, immunoreactive SQSTM1 also accumulated in the olfactory bulb of the brain. Olfactory bulb SQSTM1 often congregated in activated microglial cells that also contained olfactory marker protein, indicating neuronophagia within the olfactory bulb. This suggests the possibility that SQSTM1 or damaged proteins may be transported from the nose to the brain. Together, these findings strongly implicate widespread protein damage in the etiology of flavorings-related lung disease. |
Differential pulmonary effects of CoO and La2O3 metal oxide nanoparticle responses during aerosolized inhalation in mice
Sisler JD , Li R , McKinney W , Mercer RR , Ji Z , Xia T , Wang X , Shaffer J , Orandle M , Mihalchik AL , Battelli L , Chen BT , Wolfarth M , Andrew ME , Schwegler-Berry D , Porter DW , Castranova V , Nel A , Qian Y . Part Fibre Toxicol 2016 13 (1) 42 BACKGROUND: Although classified as metal oxides, cobalt monoxide (CoO) and lanthanum oxide (La2O3) nanoparticles, as representative transition and rare earth oxides, exhibit distinct material properties that may result in different hazardous potential in the lung. The current study was undertaken to compare the pulmonary effects of aerosolized whole body inhalation of these nanoparticles in mice. RESULTS: Mice were exposed to filtered air (control) and 10 or 30 mg/m(3) of each particle type for 4 days and then examined at 1 h, 1, 7 and 56 days post-exposure. The whole lung burden 1 h after the 4 day inhalation of CoO nanoparticles was 25 % of that for La2O3 nanoparticles. At 56 days post exposure, < 1 % of CoO nanoparticles remained in the lungs; however, 22-50 % of the La2O3 nanoparticles lung burden 1 h post exposure was retained at 56 days post exposure for low and high exposures. Significant accumulation of La2O3 nanoparticles in the tracheobronchial lymph nodes was noted at 56 days post exposure. When exposed to phagolysosomal simulated fluid, La nanoparticles formed urchin-shaped LaPO4 structures, suggesting that retention of this rare earth oxide nanoparticle may be due to complexation of cellular phosphates within lysosomes. CoO nanoparticles caused greater lactate dehydrogenase release in the bronchoalveolar fluid (BALF) compared to La2O3 nanoparticles at 1 day post exposure, while BAL cell differentials indicate that La2O3 nanoparticles generated more inflammatory cell infiltration at all doses and exposure points. Histopathological analysis showed acute inflammatory changes at 1 day after inhalation of either CoO or La2O3 nanoparticles. Only the 30 mg/m(3) La2O3 nanoparticles exposure caused chronic inflammatory changes and minimal fibrosis at day 56 post exposure. This is in agreement with activation of the NRLP3 inflammasome after in vitro exposure of differentiated THP-1 macrophages to La2O3 but not after CoO nanoparticles exposure. CONCLUSION: Taken together, the inhalation studies confirmed the trend of our previous sub-acute aspiration study, which reported that CoO nanoparticles induced more acute pulmonary toxicity, while La2O3 nanoparticles caused chronic inflammatory changes and minimal fibrosis. |
Multiwalled carbon nanotube-induced pulmonary inflammatory and fibrotic responses and genomic changes following aspiration exposure in mice: A 1-year postexposure study
Snyder-Talkington BN , Dong C , Porter DW , Ducatman B , Wolfarth MG , Andrew M , Battelli L , Raese R , Castranova V , Guo NL , Qian Y . J Toxicol Environ Health A 2016 79 (8) 1-15 Pulmonary exposure to multiwalled carbon nanotubes (MWCNT) induces an inflammatory and rapid fibrotic response, although the long-term signaling mechanisms are unknown. The aim of this study was to examine the effects of 1, 10, 40, or 80 mug MWCNT administered by pharyngeal aspiration on bronchoalveolar lavage (BAL) fluid for polymorphonuclear cell (PMN) infiltration, lactate dehydrogenase (LDH) activity, and lung histopathology for inflammatory and fibrotic responses in mouse lungs 1 mo, 6 mo, and 1 yr postexposure. Further, a 120-mug crocidolite asbestos group was incorporated as a positive control for comparative purposes. Results showed that MWCNT increased BAL fluid LDH activity and PMN infiltration in a dose-dependent manner at all three postexposure times. Asbestos exposure elevated LDH activity at all 3 postexposure times and PMN infiltration at 1 mo and 6 mo postexposure. Pathological changes in the lung, the presence of MWCNT or asbestos, and fibrosis were noted at 40 and 80 mug MWCNT and in asbestos-exposed mice at 1 yr postexposure. To determine potential signaling pathways involved with MWCNT-associated pathological changes in comparison to asbestos, up- and down-regulated gene expression was determined in lung tissue at 1 yr postexposure. Exposure to MWCNT tended to favor those pathways involved in immune responses, specifically T-cell responses, whereas exposure to asbestos tended to favor pathways involved in oxygen species production, electron transport, and cancer. Data indicate that MWCNT are biopersistent in the lung and induce inflammatory and fibrotic pathological alterations similar to those of crocidolite asbestos, but may reach these endpoints by different mechanisms. |
Common and distinct mechanisms of induced pulmonary fibrosis by particulate and soluble chemical fibrogenic agents.
Dong J , Yu X , Porter DW , Battelli LA , Kashon ML , Ma Q . Arch Toxicol 2015 90 (2) 385-402 Pulmonary fibrosis results from the excessive deposition of collagen fibers and scarring in the lungs with or without an identifiable cause. The mechanism(s) underlying lung fibrosis development is poorly understood, and effective treatment is lacking. Here we compared mouse lung fibrosis induced by pulmonary exposure to prototypical particulate (crystalline silica) or soluble chemical (bleomycin or paraquat) fibrogenic agents to identify the underlying mechanisms. Young male C57BL/6J mice were given silica (2 mg), bleomycin (0.07 mg), or paraquat (0.02 mg) by pharyngeal aspiration. All treatments induced significant inflammatory infiltration and collagen deposition, manifesting fibrotic foci in silica-exposed lungs or diffuse fibrosis in bleomycin or paraquat-exposed lungs on day 7 post-exposure, at which time the lesions reached their peaks and represented a junction of transition from an acute response to chronic fibrosis. Lung genome-wide gene expression was analyzed, and differential gene expression was confirmed by quantitative RT-PCR, immunohistochemistry, and immunoblotting for representative genes to demonstrate their induced expression and localization in fibrotic lungs. Canonical signaling pathways, gene ontology, and upstream transcription networks modified by each agent were identified. In particular, these inducers elicited marked proliferative responses; at the same time, silica preferentially activated innate immune functions and the defense against foreign bodies, whereas bleomycin and paraquat boosted responses related to cell adhesion, platelet activation, extracellular matrix remodeling, and wound healing. This study identified, for the first time, the shared and unique genes, signaling pathways, and biological functions regulated by particulate and soluble chemical fibrogenic agents during lung fibrosis, providing insights into the mechanisms underlying human lung fibrotic diseases. |
mRNAs and miRNAs in whole blood associated with lung hyperplasia, fibrosis, and bronchiolo-alveolar adenoma and adenocarcinoma after multi-walled carbon nanotube inhalation exposure in mice
Snyder-Talkington BN , Dong C , Sargent LM , Porter DW , Staska LM , Hubbs AF , Raese R , McKinney W , Chen BT , Battelli L , Lowry DT , Reynolds SH , Castranova V , Qian Y , Guo NL . J Appl Toxicol 2015 36 (1) 161-74 Inhalation exposure to multi-walled carbon nanotubes (MWCNT) in mice results in inflammation, fibrosis and the promotion of lung adenocarcinoma; however, the molecular basis behind these pathologies is unknown. This study determined global mRNA and miRNA profiles in whole blood from mice exposed by inhalation to MWCNT that correlated with the presence of lung hyperplasia, fibrosis, and bronchiolo-alveolar adenoma and adenocarcinoma. Six-week-old, male, B6C3F1 mice received a single intraperitoneal injection of either the DNA-damaging agent methylcholanthrene (MCA, 10 microg g-1 body weight) or vehicle (corn oil). One week after injections, mice were exposed by inhalation to MWCNT (5 mg m-3 , 5 hours per day, 5 days per week) or filtered air (control) for a total of 15 days. At 17 months post-exposure, mice were euthanized and examined for the development of pathological changes in the lung, and whole blood was collected and analyzed using microarray analysis for global mRNA and miRNA expression. Numerous mRNAs and miRNAs in the blood were significantly up- or down-regulated in animals developing pathological changes in the lung after MCA/corn oil administration followed by MWCNT/air inhalation, including fcrl5 and miR-122-5p in the presence of hyperplasia, mthfd2 and miR-206-3p in the presence of fibrosis, fam178a and miR-130a-3p in the presence of bronchiolo-alveolar adenoma, and il7r and miR-210-3p in the presence of bronchiolo-alveolar adenocarcinoma, among others. The changes in miRNA and mRNA expression, and their respective regulatory networks, identified in this study may potentially serve as blood biomarkers for MWCNT-induced lung pathological changes. |
Oncoprotein mdig contributes to silica-induced pulmonary fibrosis by altering balance between Th17 and Treg T cells
Thakur C , Wolfarth M , Sun J , Zhang Y , Lu Y , Battelli L , Porter DW , Chen F . Oncotarget 2015 6 (6) 3722-36 Mineral dust-induced gene (mdig, also named Mina53) was first identified from alveolar macrophages of the coal miners with chronic lung inflammation or fibrosis, but how this gene is involved in lung diseases is poorly understood. Here we show that heterozygotic knockout of mdig (mdig+/-) ameliorates silica-induced lung fibrosis by altering the balance between Th17 cells and Treg cells. Relative to the wild type (WT) mice, infiltration of the macrophages and Th17 cells was reduced in lungs from silica-exposed mdig+/- mice. In contrast, an increased infiltration of the T regulatory (Treg) cells to the lung intestitium was observed in the mdig+/- mice treated with silica. Both the number of Th17 cells in the lung lymph nodes and the level of IL-17 in the bronchoalveolar lavage fluids were decreased in the mdig+/- mice in response to silica. Thus, these results suggest that mdig may contribute to silica-induced lung fibrosis by altering the balance between Th17 and Treg cells. Genetic deficiency of mdig impairs Th17 cell infiltration and function, but favors infiltration of the Treg cells, the immune suppressive T cells that are able to limit the inflammatory responses by repressing the Th17 cells and macrophages. |
Effect of fiber length on carbon nanotube-induced fibrogenesis
Manke A , Luanpitpong S , Dong C , Wang L , He X , Battelli L , Derk R , Stueckle TA , Porter DW , Sager T , Gou H , Dinu CZ , Wu N , Mercer RR , Rojanasakul Y . Int J Mol Sci 2014 15 (5) 7444-7461 Given their extremely small size and light weight, carbon nanotubes (CNTs) can be readily inhaled by human lungs resulting in increased rates of pulmonary disorders, particularly fibrosis. Although the fibrogenic potential of CNTs is well established, there is a lack of consensus regarding the contribution of physicochemical attributes of CNTs on the underlying fibrotic outcome. We designed an experimentally validated in vitro fibroblast culture model aimed at investigating the effect of fiber length on single-walled CNT (SWCNT)-induced pulmonary fibrosis. The fibrogenic response to short and long SWCNTs was assessed via oxidative stress generation, collagen expression and transforming growth factor-beta (TGF-beta) production as potential fibrosis biomarkers. Long SWCNTs were significantly more potent than short SWCNTs in terms of reactive oxygen species (ROS) response, collagen production and TGF-beta release. Furthermore, our finding on the length-dependent in vitro fibrogenic response was validated by the in vivo lung fibrosis outcome, thus supporting the predictive value of the in vitro model. Our results also demonstrated the key role of ROS in SWCNT-induced collagen expression and TGF-beta activation, indicating the potential mechanisms of length-dependent SWCNT-induced fibrosis. Together, our study provides new evidence for the role of fiber length in SWCNT-induced lung fibrosis and offers a rapid cell-based assay for fibrogenicity testing of nanomaterials with the ability to predict pulmonary fibrogenic response in vivo. |
Promotion of lung adenocarcinoma following inhalation exposure to multi-walled carbon nanotubes
Sargent LM , Porter DW , Staska LM , Hubbs AF , Lowry DT , Battelli L , Siegrist KJ , Kashon ML , Mercer RR , Bauer AK , Chen BT , Salisbury JL , Frazer D , McKinney W , Andrew M , Tsuruoka S , Endo M , Fluharty KL , Castranova V , Reynolds SH . Part Fibre Toxicol 2014 11 (1) 3 BACKGROUND: Engineered carbon nanotubes are currently used in many consumer and industrial products such as paints, sunscreens, cosmetics, toiletries, electronic processes and industrial lubricants. Carbon nanotubes are among the more widely used nanoparticles and come in two major commercial forms, single-walled carbon nanotubes (SWCNT) and the more rigid, multi-walled carbon nanotubes (MWCNT). The low density and small size of these particles makes respiratory exposures likely. Many of the potential health hazards have not been investigated, including their potential for carcinogenicity. We, therefore, utilized a two stage initiation/promotion protocol to determine whether inhaled MWCNT act as a complete carcinogen and/or promote the growth of cells with existing DNA damage. Six week old, male, B6C3F1 mice received a single intraperitoneal (ip) injection of either the initiator methylcholanthrene(MCA, 10 mug/g BW, i.p.), or vehicle (corn oil). One week after i.p. injections, mice were exposed by inhalation to MWCNT (5 mg/m3, 5 hours/day, 5 days/week) or filtered air (controls) for a total of 15 days. At 17 months post-exposure, mice were euthanized and examined for lung tumor formation. RESULTS: Twenty-three percent of the filtered air controls, 26.5% of the MWCNT-exposed, and 51.9% of the MCA-exposed mice, had lung bronchiolo-alveolar adenomas and lung adenocarcinomas. The average number of tumors per mouse was 0.25, 0.81 and 0.38 respectively. By contrast, 90.5% of the mice which received MCA followed by MWCNT had bronchiolo-alveolar adenomas and adenocarcinomas with an average of 2.9 tumors per mouse 17months after exposure. Indeed, 62% of the mice exposed to MCA followed by MWCNT had bronchiolo-alveolar adenocarcinomas compared to 13% of the mice that received filtered air, 22% of the MCA-exposed, or 14% of the MWCNT-exposed. Mice with early morbidity resulting in euthanasia had the highest rate of metastatic disease. Three mice exposed to both MCA and MWCNT that were euthanized early had lung adenocarcinoma with evidence of metastasis (5.5%). Five mice (9%) exposed to MCA and MWCNT and 1 (1.6%) exposed to MCA developed serosal tumors morphologically consistent with sarcomatous mesotheliomas, whereas mice administered MWCNT or air alone did not develop similar neoplasms. CONCLUSIONS: These data demonstrate that some MWCNT exposures promote the growth and neoplastic progression of initiated lung cells in B6C3F1 mice. In this study, the mouse MWCNT lung burden of 31.2 mug/mouse approximates feasible human occupational exposures. Therefore, the results of this study indicate that caution should be used to limit human exposures to MWCNT. |
Carbon nanotube dosimetry: from workplace exposure assessment to inhalation toxicology
Erdely A , Dahm M , Chen BT , Zeidler-Erdely PC , Fernback JE , Birch ME , Evans DE , Kashon ML , Deddens JA , Hulderman T , Bilgesu SA , Battelli L , Schwegler-Berry D , Leonard HD , McKinney W , Frazer DG , Antonini JM , Porter DW , Castranova V , Schubauer-Berigan MK . Part Fibre Toxicol 2013 10 (1) 53 BACKGROUND: Dosimetry for toxicology studies involving carbon nanotubes (CNT) is challenging because of a lack of detailed occupational exposure assessments. Therefore, exposure assessment findings, measuring the mass concentration of elemental carbon from personal breathing zone (PBZ) samples, from 8 U.S.-based multi-walled CNT (MWCNT) manufacturers and users were extrapolated to results of an inhalation study in mice. RESULTS: Upon analysis, an inhalable elemental carbon mass concentration arithmetic mean of 10.6 mug/m3 (geometric mean 4.21 mug/m3) was found among workers exposed to MWCNT. The concentration equates to a deposited dose of approximately 4.07 mug/d in a human, equivalent to 2 ng/d in the mouse. For MWCNT inhalation, mice were exposed for 19 d with daily depositions of 1970 ng (equivalent to 1000 d of a human exposure; cumulative 76 yr), 197 ng (100 d; 7.6 yr), and 19.7 ng (10 d; 0.76 yr) and harvested at 0, 3, 28, and 84 d post-exposure to assess pulmonary toxicity. The high dose showed cytotoxicity and inflammation that persisted through 84 d after exposure. The middle dose had no polymorphonuclear cell influx with transient cytotoxicity. The low dose was associated with a low grade inflammatory response measured by changes in mRNA expression. Increased inflammatory proteins were present in the lavage fluid at the high and middle dose through 28 d post-exposure. Pathology, including epithelial hyperplasia and peribronchiolar inflammation, was only noted at the high dose. CONCLUSION: These findings showed a limited pulmonary inflammatory potential of MWCNT at levels corresponding to the average inhalable elemental carbon concentrations observed in U.S.-based CNT facilities and estimates suggest considerable years of exposure are necessary for significant pathology to occur at that level. |
Investigation of the pulmonary bioactivity of double-walled carbon nanotubes
Sager TM , Wolfarth MW , Battelli LA , Leonard SS , Andrew M , Steinbach T , Endo M , Tsuruoka S , Porter DW , Castranova V . J Toxicol Environ Health A 2013 76 (15) 922-36 Double-walled carbon nanotubes (DWCNT) are a rather new and unexplored variety of carbon nanotubes. Previously conducted studies established that exposure to a variety of carbon nanotubes produced lung inflammation and fibrosis in mice after pharyngeal aspiration. However, the bioactivity of double-walled carbon nanotubes (DWCNT) has not been determined. In this study, the hypothesis that DWCNT would induce pulmonary toxicity was explored by analyzing the pulmonary bioactivity of DWCNT. To test this hypothesis, C57Bl/6 mice were exposed to DWCNT by pharyngeal aspiration. Mice underwent whole-lung lavage (WLL) to assess pulmonary inflammation and injury, and lung tissue was examined histologically for development of pulmonary disease as a function of dose and time. The results showed that DWCNT exposure produced a dose-dependent increase in WLL polymorphonuclear leukocytes (PMN), indicating that DWCNT exposure initiated pulmonary inflammation. DWCNT exposure also produced a dose-dependent rise in lactate dehydrogenase (LDH) activity, as well as albumin levels, in WLL fluid, indicating that DWCNT exposure promoted cytotoxicity as well as decreases in the integrity of the blood-gas barrier in the lung, respectively. In addition, at 7 and 56 d postexposure, the presence of significant alveolitis and fibrosis was noted in mice exposed to 40 mug/mouse DWCNT. In conclusion, this study provides insight into previously uninvestigated pulmonary bioactivity of DWCNT exposure. Data indicate that DWCNT exposure promotes inflammation, injury, and fibrosis in the lung. |
Lung tumor promotion by chromium-containing welding particulate matter in a mouse model
Zeidler-Erdely PC , Meighan TG , Erdely A , Battelli LA , Kashon ML , Keane M , Antonini JM . Part Fibre Toxicol 2013 10 (1) 45 BACKGROUND: Epidemiology suggests that occupational exposure to welding particulate matter (PM) may increase lung cancer risk. However, animal studies are lacking to conclusively link welding with an increased risk. PM derived from stainless steel (SS) welding contains carcinogenic metals such as hexavalent chromium and nickel. We hypothesized that welding PM may act as a tumor promoter and increase lung tumor multiplicity in vivo. Therefore, the capacity of chromium-containing gas metal arc (GMA)-SS welding PM to promote lung tumors was evaluated using a two-stage (initiation-promotion) model in lung tumor susceptible A/J mice. METHODS: Male mice (n = 28-30/group) were treated either with the initiator 3-methylcholanthrene (MCA;10 microg/g; IP) or vehicle (corn oil) followed by 5 weekly pharyngeal aspirations of GMA-SS (340 or 680 microg/exposure) or PBS. Lung tumors were enumerated at 30 weeks post-initiation. RESULTS: MCA initiation followed by GMA-SS welding PM exposure promoted tumor multiplicity in both the low (12.1 = 1.5 tumors/mouse) and high (14.0 +/- 1.8 tumors/mouse) exposure groups significantly above MCA/sham (4.77 +/- 0.7 tumors/mouse; p = 0.0001). Multiplicity was also highly significant (p < 0.004) across all individual lung regions of GMA-SS-exposed mice. No exposure effects were found in the corn oil groups at 30 weeks. Histopathology confirmed the gross findings and revealed increased inflammation and a greater number of malignant lesions in the MCA/welding PM-exposed groups. CONCLUSIONS: GMA-SS welding PM acts as a lung tumor promoter in vivo. Thus, this study provides animal evidence to support the epidemiological data that show welders have an increased lung cancer risk. |
Extrapulmonary transport of MWCNT following inhalation exposure
Mercer RR , Scabilloni JF , Hubbs AF , Wang L , Battelli LA , McKinney W , Castranova V , Porter DW . Part Fibre Toxicol 2013 10 (1) 38 BACKGROUND: Inhalation exposure studies of mice were conducted to determine if multi-walled carbon nanotubes (MWCNT) distribute to the tracheobronchial lymphatics, parietal pleura, respiratory musculature and/or extrapulmonary organs. Male C57BL/6 J mice were exposed in a whole-body inhalation system to a 5 mg/m3 MWCNT aerosol for 5 hours/day for 12 days (4 times/week for 3 weeks, lung burden 28.1 ug/lung). At 1 day and 336 days after the 12 day exposure period, mice were anesthetized and lungs, lymph nodes and extrapulmonary tissues were preserved by whole body vascular perfusion of paraformaldehyde while the lungs were inflated with air. Separate, clean-air control groups were studied at 1 day and 336 days post-exposure. Sirius Red stained sections from lung, tracheobronchial lymph nodes, diaphragm, chest wall, heart, brain, kidney and liver were analyzed. Enhanced darkfield microscopy and morphometric methods were used to detect and count MWCNT in tissue sections. Counts in tissue sections were expressed as number of MWCNT per g of tissue and as a percentage of total lung burden (Mean +/- S.E., N = 8 mice per group). MWCNT burden in tracheobronchial lymph nodes was determined separately based on the volume density in the lymph nodes relative to the volume density in the lungs. Field emission scanning electron microscopy (FESEM) was used to examine MWCNT structure in the various tissues. RESULTS: Tracheobronchial lymph nodes were found to contain 1.08 and 7.34 percent of the lung burden at 1 day and 336 days post-exposure, respectively. Although agglomerates account for approximately 54% of lung burden, only singlet MWCNT were observed in the diaphragm, chest wall, liver, kidney, heart and brain. At one day post exposure, the average length of singlet MWCNT in liver and kidney, was comparable to that of singlet MWCNT in the lungs 8.2 +/- 0.3 versus 7.5 +/- 0.4 um, respectively. On average, there were 15,371 and 109,885 fibers per gram in liver, kidney, heart and brain at 1 day and 336 days post-exposure, respectively. The burden of singlet MWCNT in the lymph nodes, diaphragm, chest wall and extrapulmonary organs at 336 days post-exposure was significantly higher than at 1 day post-exposure. CONCLUSIONS: Inhaled MWCNT, which deposit in the lungs, are transported to the parietal pleura, the respiratory musculature, liver, kidney, heart and brain in a singlet form and accumulate with time following exposure. The tracheobronchial lymph nodes contain high levels of MWCNT following exposure and further accumulate over nearly a year to levels that are a significant fraction of the lung burden 1 day post-exposure. |
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