Last data update: Apr 29, 2024. (Total: 46658 publications since 2009)
Records 1-30 (of 33 Records) |
Query Trace: Wolfarth M [original query] |
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Potent lung tumor promotion by inhaled MWCNT
Porter DW , Orandle MS , Hubbs A , Staska LM , Lowry D , Kashon M , Wolfarth MG , McKinney W , Sargent LM . Nanotoxicology 2024 1-18 In the lung, carcinogenesis is a multi-stage process that includes initiation by a genotoxic agent, promotion that expands the population of cells with damaged DNA to form a tumor, and progression from benign to malignant neoplasms. We have previously shown that Mitsui-7, a long and rigid multi-walled carbon nanotube (MWCNT), promotes pulmonary carcinogenesis in a mouse model. To investigate the potential exposure threshold and dose-response for tumor promotion by this MWCNT, 3-methylcholanthrene (MC) initiated (10 μg/g, i.p., once) or vehicle (corn oil) treated B6C3F1 mice were exposed by inhalation to filtered air or MWCNT (5 mg/m(3)) for 5 h/day for 0, 2, 5, or 10 days and were followed for 17 months post-exposure for evidence of lung tumors. Pulmonary neoplasia incidence in MC-initiated mice significantly increased with each MWCNT exposure duration. Exposure to either MC or MWCNT alone did not affect pulmonary neoplasia incidence compared with vehicle controls. Lung tumor multiplicity in MC-initiated mice also significantly increased with each MWCNT exposure duration. Thus, a significantly higher lung tumor multiplicity was observed after a 10-day MWCNT exposure than following a 2-day exposure. Both bronchioloalveolar adenoma and bronchioloalveolar adenocarcinoma multiplicity in MC-initiated mice were significantly increased following 5- and 10-day MWCNT exposure, while a 2-day MWCNT exposure in MC-initiated mice significantly increased the multiplicity of adenomas but not adenocarcinomas. In this study, even the lowest MWCNT exposure promoted lung tumors in MC-initiated mice. Our findings indicate that exposure to this MWCNT strongly promotes pulmonary carcinogenesis. |
Influence of impurities from manufacturing process on the toxicity profile of boron nitride nanotubes
Kodali V , Kim KS , Roberts JR , Bowers L , Wolfarth MG , Hubczak J , Xin X , Eye T , Friend S , Stefaniak AB , Leonard SS , Jakubinek M , Erdely A . Small 2022 18 (52) e2203259 The toxicity of boron nitride nanotubes (BNNTs) has been the subject of conflicting reports, likely due to differences in the residuals and impurities that can make up to 30-60% of the material produced based on the manufacturing processes and purification employed. Four BNNTs manufactured by induction thermal plasma process with a gradient of BNNT purity levels achieved through sequential gas purification, water and solvent washing, allowed assessing the influence of these residuals/impurities on the toxicity profile of BNNTs. Extensive characterization including infrared and X-ray spectroscopy, thermogravimetric analysis, size, charge, surface area, and density captured the alteration in physicochemical properties as the material went through sequential purification. The material from each step is screened using acellular and in vitro assays for evaluating general toxicity, mechanisms of toxicity, and macrophage function. As the material increased in purity, there are more high-aspect-ratio particulates and a corresponding distinct increase in cytotoxicity, nuclear factor-κB transcription, and inflammasome activation. There is no alteration in macrophage function after BNNT exposure with all purity grades. The cytotoxicity and mechanism of screening clustered with the purity grade of BNNTs, illustrating that greater purity of BNNT corresponds to greater toxicity. |
Developing a solution for nasal and olfactory transport of nanomaterials
O'Connell RC , Dodd TM , Clingerman SM , Fluharty KL , Coyle J , Stueckle TA , Porter DW , Bowers L , Stefaniak AB , Knepp AK , Derk R , Wolfarth M , Mercer RR , Boots TE , Sriram K , Hubbs AF . Toxicol Pathol 2022 50 (3) 1926233221089209 With advances in nanotechnology, engineered nanomaterial applications are a rapidly growing sector of the economy. Some nanomaterials can reach the brain through nose-to-brain transport. This transport creates concern for potential neurotoxicity of insoluble nanomaterials and a need for toxicity screening tests that detect nose-to-brain transport. Such tests can involve intranasal instillation of aqueous suspensions of nanomaterials in dispersion media that limit particle agglomeration. Unfortunately, protein and some elements in existing dispersion media are suboptimal for potential nose-to-brain transport of nanomaterials because olfactory transport has size- and ion-composition requirements. Therefore, we designed a protein-free dispersion media containing phospholipids and amino acids in an isotonic balanced electrolyte solution, a solution for nasal and olfactory transport (SNOT). SNOT disperses hexagonal boron nitride nanomaterials with a peak particle diameter below 100 nm. In addition, multiwalled carbon nanotubes (MWCNTs) in an established dispersion medium, when diluted with SNOT, maintain dispersion with reduced albumin concentration. Using stereomicroscopy and microscopic examination of plastic sections, dextran dyes dispersed in SNOT are demonstrated in the neuroepithelium of the nose and olfactory bulb of B6;129P2-Omp(tm3Mom)/MomJ mice after intranasal instillation in SNOT. These findings support the potential for SNOT to disperse nanomaterials in a manner permitting nose-to-brain transport for neurotoxicity studies. |
Toxicity evaluation following pulmonary exposure to an as-manufactured dispersed boron nitride nanotube (BNNT) material in vivo
Xin X , Barger M , Roach KA , Bowers L , Stefaniak AB , Kodali V , Glassford E , Dunn KL , Dunn KH , Wolfarth M , Friend S , Leonard SS , Kashon M , Porter DW , Erdely A , Roberts JR . NanoImpact 2020 19 Boron nitride nanotubes (BNNT) are multi-walled nanotubes composed of hexagonal B[sbnd]N bonds and possess many unique physical and chemical properties, creating a rapidly expanding market for this newly emerging nanomaterial which is still primarily in the research and development stage. The shape and high aspect ratio give rise to concern for the potential toxicity that may be associated with pulmonary exposure, especially in an occupational setting. The goal of this study was to assess lung toxicity using an in vivo time course model. The sample was manufactured to be 5 nm wide and up to 200 μm long, with ~50% purity covalently bound with hexagonal boron nitride (hBN) in the sample. Following preparation for in vivo studies, sonication of the material disrupted the longer tubes in the complex and the size distribution in dispersion medium (DM) of the structures was 13–23 nm in diameter and 0.6–1.6 μm in length. Male C57BL/6 J mice were exposed to 4 or 40 μg of BNNT or DM (vehicle control) by a single oropharyngeal aspiration. Pulmonary and systemic toxicity were investigated at 4 h, 1 d, 7 d, 1 mo and 2 mo post-exposure. Bronchoalveolar lavage (BAL) studies determined pulmonary inflammation (neutrophil influx) and cytotoxicity (lactate dehydrogenase activity) occurred at early time points and peaked at 7 d post-exposure in the high dose group. Histopathological analysis showed a minimal level of inflammatory cell infiltration in the high dose group with resolution over time and no fibrosis, and lung clearance analysis showed ~50% of the material cleared over the time course. The expression of inflammatory- and acute phase response-associated genes in the lung and liver were significantly increased by the high dose at 4 h and 1 d post-exposure. The increases in lung gene expression of Cxcl2, Ccl2, Il6, Ccl22, Ccl11, and Spp1 were significant up to 2 mo but decreased with time. The low dose exposure did not result in significant changes in any toxicological parameters measured. In summary, the BNNT-hBN sample used in this study caused acute pulmonary inflammation and injury at the higher dose, which peaked by 7 d post-exposure and showed resolution over time. Further studies are needed to determine if physicochemical properties and purity will impact the toxicity profile of BNNT and to investigate the underlying mechanisms of BNNT toxicity. |
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. |
Multi-Walled Carbon Nanotube-Induced Gene Expression Biomarkers for Medical and Occupational Surveillance.
Snyder-Talkington BN , Dong C , Singh S , Raese R , Qian Y , Porter DW , Wolfarth MG , Guo NL . Int J Mol Sci 2019 20 (11) As the demand for multi-walled carbon nanotube (MWCNT) incorporation into industrial and biomedical applications increases, so does the potential for unintentional pulmonary MWCNT exposure, particularly among workers during manufacturing. Pulmonary exposure to MWCNTs raises the potential for development of lung inflammation, fibrosis, and cancer among those exposed; however, there are currently no effective biomarkers for detecting lung fibrosis or predicting the risk of lung cancer resulting from MWCNT exposure. To uncover potential mRNAs and miRNAs that could be used as markers of exposure, this study compared in vivo mRNA and miRNA expression in lung tissue and blood of mice exposed to MWCNTs with in vitro mRNA and miRNA expression from a co-culture model of human lung epithelial and microvascular cells, a system previously shown to have a higher overall genome-scale correlation with mRNA expression in mouse lungs than either cell type grown separately. Concordant mRNAs and miRNAs identified by this study could be used to drive future studies confirming human biomarkers of MWCNT exposure. These potential biomarkers could be used to assess overall worker health and predict the occurrence of MWCNT-induced diseases. |
Acute in vitro and in vivo toxicity of a commercial grade boron nitride nanotube mixture
Kodali VK , Roberts JR , Shoeb M , Wolfarth MG , Bishop L , Eye T , Barger M , Roach KA , Friend S , Schwegler-Berry D , Chen BT , Stefaniak A , Jordan KC , Whitney RR , Porter DW , Erdely AD . Nanotoxicology 2017 11 (8) 1-19 Boron nitride nanotubes (BNNTs) are an emerging engineered nanomaterial attracting significant attention due to superior electrical, chemical and thermal properties. Currently, the toxicity profile of this material is largely unknown. Commercial grade BNNTs are composed of a mixture (BNNT-M) of approximately 50-60% BNNTs, and approximately 40-50% impurities of boron and hexagonal boron nitride. We performed acute in vitro and in vivo studies with commercial grade BNNT-M, dispersed by sonication in vehicle, in comparison to the extensively studied multiwalled carbon nanotube-7 (MWCNT-7). THP-1 wild-type and NLRP3-deficient human monocytic cells were exposed to 0-100 microg/ml and C57BL/6 J male mice were treated with 40 microg of BNNT-M for in vitro and in vivo studies, respectively. In vitro, BNNT-M induced a dose-dependent increase in cytotoxicity and oxidative stress. This was confirmed in vivo following acute exposure increase in bronchoalveolar lavage levels of lactate dehydrogenase, pulmonary polymorphonuclear cell influx, loss in mitochondrial membrane potential and augmented levels of 4-hydroxynonenal. Uptake of this material caused lysosomal destabilization, pyroptosis and inflammasome activation, corroborated by an increase in cathepsin B, caspase 1, increased protein levels of IL-1beta and IL-18 both in vitro and in vivo. Attenuation of these effects in NLRP3-deficient THP-1 cells confirmed NLRP3-dependent inflammasome activation by BNNT-M. BNNT-M induced a similar profile of inflammatory pulmonary protein production when compared to MWCNT-7. Functionally, pretreatment with BNNT-M caused suppression in bacterial uptake by THP-1 cells, an effect that was mirrored in challenged alveolar macrophages collected from exposed mice and attenuated with NLRP3 deficiency. Analysis of cytokines secreted by LPS-challenged alveolar macrophages collected after in vivo exposure to dispersions of BNNT-M showed a differential macrophage response. The observed results demonstrated acute inflammation and toxicity in vitro and in vivo following exposure to sonicated BNNT-M was in part due to NLRP3 inflammasome activation. |
Length, but not reactive edges, of cup-stack MWCNT is responsible for toxicity and acute lung inflammation
Hamilton RF Jr , Tsuruoka S , Wu N , Wolfarth M , Porter DW , Bunderson-Schelvan M , Holian A . Toxicol Pathol 2017 46 (1) 192623317732303 Multiwalled carbon nanotube (MWCNT) toxicity after inhalation has been associated with size, aspect ratio, rigidity, surface modification, and reactive oxygen species production. In this study, we investigated a series of cup-stacked MWCNT prepared as variants of the Creos 24PS. Mechanical chopping produced a short version (AR10) and graphitization to remove active reaction sites by extreme heat (2,800 degrees C; Creos 24HT) to test the contribution of length and alteration of potential reaction sites to toxicity. The 3 MWCNT variants were tested in vitro in a human macrophage-like cell model and with C57BL/6 alveolar macrophages for dose-dependent toxicity and NLRP3 inflammasome activation. The 24PS and 24HT variants showed significant dose-dependent toxicity and inflammasome activation. In contrast, the AR10 variant showed no toxicity or bioactivity at any concentration tested. The in vivo results reflected those observed in vitro, with the 24PS and 24HT variants resulting in acute inflammation, including elevated polymorphonuclear counts, Interleukin (IL)-18, cathepsin B, and lactate dehydrogenase in isolated lung lavage fluid from mice exposed to 40 microg MWCNT. Taken together, these data indicate that length, but not the absence of proposed reaction sites, on the MWCNT influences particle bioactivity. |
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. |
Role of engineered metal oxide nanoparticle agglomeration in reactive oxygen species generation and cathepsin B release in NLRP3 inflammasome activation and pulmonary toxicity
Sager TM , Wolfarth M , Leonard SS , Morris AM , Porter DW , Castranova V , Holian A . Inhal Toxicol 2016 28 (14) 1-12 Incomplete understanding of the contributions of dispersants and engineered nanoparticles/materials (ENM) agglomeration state to biological outcomes presents an obstacle for toxicological studies. Although reactive oxygen species (ROS) production is often regarded as the primary indicator of ENM bioactivity and toxicity, it remains unclear whether ENM produce ROS or whether ROS is an outcome of ENM-induced cell injury. Phagolysosomal disruption and cathepsin B release also promote bioactivity through inflammasome activation. Therefore, specific particle parameters, i.e. preexposure dispersion status and particle surface area, of two ENM (NiO and CeO2) were used to evaluate the role of ROS generation and cathepsin B release during ENM-induced toxicity. Male C57BL/6J mice were exposed to 0, 20, 40, or 80 mug of poorly or well-dispersed NiO-NP or CeO2-NP in four types of dispersion media. At 1- and 7-day postexposure, lung lavage fluid was collected to assess inflammation, cytotoxicity, and inflammasome activation. Results showed that preexposure dispersion status correlated with postexposure pulmonary bioactivity. The differences in bioactivity of NiO-NP and CeO2-NP are likely due to NiO-NP facilitating the release of cathepsin B and in turn inflammasome activation generating proinflammatory cytokines. Further, both metal oxides acted as free radical scavengers. Depending on the pH, CeO2-NP acted as a free radical scavenger in an acidic environment (an environment mimicking the lysosome) while the NiO-NP acted as a scavenger in a physiological pH (an environment that mimics the cytosol of the cell). Therefore, results from this study suggest that ENM-induced ROS is not likely a mechanism of inflammasome activation. |
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. |
Evaluation of pulmonary and systemic toxicity following lung exposure to graphite nanoplates: A member of the graphene-based nanomaterial family
Roberts JR , Mercer RR , Stefaniak AB , Seehra MS , Geddam UK , Chaudhuri IS , Kyrlidis A , Kodali VK , Sager T , Kenyon A , Bilgesu SA , Eye T , Scabilloni JF , Leonard SS , Fix NR , Schwegler-Berry D , Farris BY , Wolfarth MG , Porter DW , Castranova V , Erdely A . Part Fibre Toxicol 2016 13 (1) 34 BACKGROUND: Graphene, a monolayer of carbon, is an engineered nanomaterial (ENM) with physical and chemical properties that may offer application advantages over other carbonaceous ENMs, such as carbon nanotubes (CNT). The goal of this study was to comparatively assess pulmonary and systemic toxicity of graphite nanoplates, a member of the graphene-based nanomaterial family, with respect to nanoplate size. METHODS: Three sizes of graphite nanoplates [20 mum lateral (Gr20), 5 mum lateral (Gr5), and <2 mum lateral (Gr1)] ranging from 8-25 nm in thickness were characterized for difference in surface area, structure,, zeta potential, and agglomeration in dispersion medium, the vehicle for in vivo studies. Mice were exposed by pharyngeal aspiration to these 3 sizes of graphite nanoplates at doses of 4 or 40 mug/mouse, or to carbon black (CB) as a carbonaceous control material. At 4 h, 1 day, 7 days, 1 month, and 2 months post-exposure, bronchoalveolar lavage was performed to collect fluid and cells for analysis of lung injury and inflammation. Particle clearance, histopathology and gene expression in lung tissue were evaluated. In addition, protein levels and gene expression were measured in blood, heart, aorta and liver to assess systemic responses. RESULTS: All Gr samples were found to be similarly composed of two graphite structures and agglomerated to varying degrees in DM in proportion to the lateral dimension. Surface area for Gr1 was approximately 7-fold greater than Gr5 and Gr20, but was less reactive reactive per m(2). At the low dose, none of the Gr materials induced toxicity. At the high dose, Gr20 and Gr5 exposure increased indices of lung inflammation and injury in lavage fluid and tissue gene expression to a greater degree and duration than Gr1 and CB. Gr5 and Gr20 showed no or minimal lung epithelial hypertrophy and hyperplasia, and no development of fibrosis by 2 months post-exposure. In addition, the aorta and liver inflammatory and acute phase genes were transiently elevated in Gr5 and Gr20, relative to Gr1. CONCLUSIONS: Pulmonary and systemic toxicity of graphite nanoplates may be dependent on lateral size and/or surface reactivity, with the graphite nanoplates > 5 mum laterally inducing greater toxicity which peaked at the early time points post-exposure relative to the 1-2 mum graphite nanoplate. |
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. |
Effects of nickel-oxide nanoparticle pre-exposure dispersion status on bioactivity in the mouse lung
Sager T , Wolfarth M , Keane M , Porter D , Castranova V , Holian A . Nanotoxicology 2016 10 (2) 151-61 Nanotechnology is emerging as one of the world's most promising new technologies. From a toxicology perspective, nanoparticles possess two features that promote their bioactivity. The first involves physical-chemical characteristics of the nanoparticle, which include the surface area of the nanoparticle. The second feature is the ability of the nanoparticle to traverse cell membranes. These two important nanoparticle characteristics are greatly influenced by placing nanoparticles in liquid medium prior to animal exposure. Nanoparticles tend to agglomerate and clump in suspension, making it difficult to reproducibly deliver them for in vivo or in vitro experiments, possibly affecting experimental variability. Thus, we hypothesize that nanoparticle dispersion status will correlate with the in vivo bioactivity/toxicity of the particle. To test our hypothesis, nano-sized nickel oxide was suspended in four different dispersion media (phosphate-buffered saline (PBS), dispersion medium (DM), a combination of dipalmitoyl-phosphatidyl choline (DPPC) and albumin in concentrations that mimic diluted alveolar lining fluid), Survanta(R), or pluronic (Pluronic F-68). Well-dispersed and poorly dispersed suspensions were generated in each media by varying sonication time on ice utilizing a Branson Sonifer 450 (25W continuous output, 20 min or 5 min, respectively). Mice (male, C57BL/6J, 7-weeks-old) were given 0-80 microg/mouse of nano-sized nickel oxide in the different states of dispersion via pharyngeal aspiration. At 1 and 7 d post-exposure, mice underwent whole lung lavage to assess pulmonary inflammation and injury as a function of dispersion status, dose and time. The results show that pre-exposure dispersion status correlates with pulmonary inflammation and injury. These results indicate that a greater degree of pre-exposure dispersion increases pulmonary inflammation and cytotoxicity, as well as decreases in the integrity of the blood-gas barrier in the lung. |
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. |
Multi-walled carbon nanotube-induced gene expression in vitro: concordance with in vivo studies
Snyder-Talkington BN , Dong C , Zhao X , Dymacek J , Porter DW , Wolfarth MG , Castranova V , Qian Y , Guo NL . Toxicology 2014 328c 66-74 There is a current interest in reducing the in vivo toxicity testing of nanomaterials in animals by increasing toxicity testing using in vitro cellular assays; however, toxicological results are seldom concordant between in vivo and in vitro models. This study compared global multi-walled carbon nanotube (MWCNT)-induced gene expression from human lung epithelial and microvascular endothelial cells in monoculture and coculture with gene expression from mouse lungs exposed to MWCNT. Using a cutoff of 10% false discovery rate and 1.5 fold change, we determined that there were more concordant genes (gene expression both up- or downregulated in vivo and in vitro) expressed in both cell types in coculture than in monoculture. When reduced to only those genes involved in inflammation and fibrosis, known outcomes of in vivo MWCNT exposure, there were more disease-related concordant genes expressed in coculture than monoculture. Additionally, different cellular signaling pathways are activated in response to MWCNT dependent upon culturing conditions. As coculture gene expression better correlated with in vivo gene expression, we suggest that cellular cocultures may offer enhanced in vitro models for nanoparticle risk assessment and the reduction of in vivo toxicological testing. |
Pathologic and molecular profiling of rapid-onset fibrosis and inflammation induced by multi-walled carbon nanotubes.
Dong J , Porter DW , Batteli LA , Wolfarth MG , Richardson DL , Ma Q . Arch Toxicol 2014 89 (4) 621-33 Multi-walled carbon nanotubes (MWCNT) are new materials with a wide range of industrial and commercial applications. However, their nano-scaled size and fiber-like shape render them respirable and potentially fibrogenic if inhaled into the lungs. To understand MWCNT fibrogenesis, we analyzed the pathologic and molecular aspects of the early phase response to MWCNT in mouse lungs. MWCNT induced rapid and pronounced lesions in the lungs characterized by increased cellularity and formation of fibrotic foci, most notably near where MWCNT deposited, within 14 days post-exposure. Deposition of collagen fibers was markedly increased in the alveolar septa and fibrotic foci, accompanied by elevated expression of fibrotic genes Col1a1, Col1a2, and Fn1 at both mRNA and protein levels. Fibrosis was induced rapidly at 40 mug, wherein fibrotic changes were detected on day 1 and reached a maximal intensity on day 7 through day 14. Induction of fibrosis was dose-dependent at the dose range of 5-40 mug, 7 days post-exposure. MWCNT elicited rapid and prominent infiltrations of neutrophils and macrophages alongside fibrosis implicating acute inflammation in the fibrotic response. At the molecular level, MWCNT induced elevated expression of proinflammatory cytokines TNFalpha, IL1alpha, IL1beta, IL6, and CCL2 in lung tissues as well as the bronchoalveolar lavage fluid, in a dose- and time-dependent manner. MWCNT also increased the expression of fibrogenic growth factors TGF-beta1 and PDGF-A in the lungs significantly. These findings underscore the interplay between acute inflammation and the early fibrotic response in the initiation and propagation of pulmonary fibrosis induced by MWCNT. |
mRNA and miRNA regulatory networks reflective of multi-walled carbon nanotube-induced lung inflammatory and fibrotic pathologies in mice.
Dymacek J , Snyder-Talkington BN , Porter DW , Wolfarth MG , Castranova V , Qian Y , Guo NL . Toxicol Sci 2014 144 (1) 51-64 Multi-walled carbon nanotubes (MWCNT) are known for their transient inflammatory and progressive fibrotic pulmonary effects; however, the mechanisms underlying these pathologies are unknown. In this study, we used time-series microarray data of global lung mRNA and miRNA expression isolated from C57BL/6J mice exposed by pharyngeal aspiration to vehicle or 10, 20, 40, or 80 mug MWCNT at 1, 7, 28, or 56 days post-exposure to determine miRNA andmRNA regulatory networks that are potentially involved in MWCNT-induced inflammatory and fibrotic lung etiology. Using a non-negative matrix factorization method, we determined mRNAs and miRNAs with expression profiles associated with pathology patterns of MWCNT-induced inflammation (based upon bronchoalveolar lavage score) and fibrosis (based upon Sirius Red staining measured with quantitative morphometric analysis). Potential binding targets between pathology-related mRNAs and miRNAs were identified using Ingenuity Pathway Analysis and the miRTarBase, miRecords, and TargetScan databases. Using these experimentally validated and predicted binding targets, we were able to build molecular signaling networks that are potentially reflective of and play a role in MWCNT-induced lung inflammatory and fibrotic pathology. As understanding the regulatory networks between mRNAs and miRNAs in different disease states would be beneficial for understanding the complex mechanisms of pathogenesis, these identified genes and pathways may be useful for determining biomarkers of MWCNT-induced lung inflammation and fibrosis for early detection of disease. |
Synthesis, characterization, and bioactivity of carboxylic acid-functionalized titanium dioxide nanobelts
Hamilton RF , Wu N , Xiang C , Li M , Yang F , Wolfarth M , Porter DW , Holian A . Part Fibre Toxicol 2014 11 (1) 43 BACKGROUND: Surface modification strategies to reduce engineered nanomaterial (ENM) bioactivity have been used successfully in carbon nanotubes. This study examined the toxicity and inflammatory potential for two surface modifications (humic acid and carboxylation) on titanium nanobelts (TNB). METHODS: The in vitro exposure models include C57BL/6 alveolar macrophages (AM) and transformed human THP-1 cells exposed to TNB for 24 hrs in culture. Cell death and NLRP3 inflammasome activation (IL-1beta release) were monitored. Short term (4 and 24 hr) in vivo studies in C57BL/6, BALB/c and IL-1R null mice evaluated inflammation and cytokine release, and cytokine release from ex vivo cultured AM. RESULTS: Both in vitro cell models suggest that the humic acid modification does not significantly affect TNB bioactivity, while carboxylation reduced both toxicity and NLRP3 inflammasome activation. In addition, short term in vivo exposures in both C57BL/6 and IL-1R null mouse strains demonstrated decreased markers of inflammation, supporting the in vitro finding that carboxylation is effective in reducing bioactivity. TNB instillations in IL-1R null mice demonstrated the critical role of IL-1beta in initiation of TNB-induced lung inflammation. Neutrophils were completely absent in the lungs of IL-1R null mice instilled with TNB for 24 hrs. However, the cytokine content of the IL-1R null mice lung lavage samples indicated that other inflammatory agents, IL-6 and TNF-alpha were constitutively elevated indicating a potential compensatory inflammatory mechanism in the absence of IL-1 receptors. CONCLUSIONS: Taken together, the data suggests that carboxylation, but not humic acid modification of TNB reduces, but does not totally eliminate bioactivity of TNB, which is consistent with previous studies of other long aspect ratio nanomaterials such as carbon nanotubes. |
Effect of multi-walled carbon nanotube surface modification on bioactivity in the C57BL/6 mouse model
Sager TM , Wolfarth MW , Andrew M , Hubbs A , Friend S , Chen TH , Porter DW , Wu N , Yang F , Hamilton RF , Holian A . Nanotoxicology 2014 8 (3) 317-27 The current study tests the hypothesis that multi-walled carbon nanotubes (MWCNT) with different surface chemistries exhibit different bioactivity profiles in vivo. In addition, the study examined the potential contribution of the NLRP3 inflammasome in MWCNT-induced lung pathology. Unmodified (BMWCNT) and MWCNT that were surface functionalised with -COOH (FMWCNT), were instilled into C57BL/6 mice. The mice were then examined for biomarkers of inflammation and injury, as well as examined histologically for development of pulmonary disease as a function of dose and time. Biomarkers for pulmonary inflammation included cytokines, mediators and the presence of inflammatory cells (IL-1beta, IL-18, IL-33, cathepsin B and neutrophils) and markers of injury (albumin and lactate dehydrogenase). The results show that surface modification by the addition of the -COOH group to the MWCNT, significantly reduced the bioactivity and pathogenicity. The results of this study also suggest that in vivo pathogenicity of the BMWCNT and FMWCNT correlates with activation of the NLRP3 inflammasome in the lung. |
Pulmonary cerium dioxide nanoparticle exposure differentially impairs coronary and mesenteric arteriolar reactivity
Minarchick VC , Stapleton PA , Porter DW , Wolfarth MG , Ciftyurek E , Barger M , Sabolsky EM , Nurkiewicz TR . Cardiovasc Toxicol 2013 13 (4) 323-37 Cerium dioxide nanoparticles (CeO2 NPs) are an engineered nanomaterial (ENM) that possesses unique catalytic, oxidative, and reductive properties. Currently, CeO2 NPs are being used as a fuel catalyst but these properties are also utilized in the development of potential drug treatments for radiation and stroke protection. These uses of CeO2 NPs present a risk for human exposure; however, to date, no studies have investigated the effects of CeO2 NPs on the microcirculation following pulmonary exposure. Previous studies in our laboratory with other nanomaterials have shown impairments in normal microvascular function after pulmonary exposures. Therefore, we predicted that CeO2 NP exposure would cause microvascular dysfunction that is dependent on the tissue bed and dose. Twenty-four-hour post-exposure to CeO2 NPs (0-400 mug), mesenteric, and coronary arterioles was isolated and microvascular function was assessed. Our results provided evidence that pulmonary CeO2 NP exposure impairs endothelium-dependent and endothelium-independent arteriolar dilation in a dose-dependent manner. The CeO2 NP exposure dose which causes a 50 % impairment in arteriolar function (EC50) was calculated and ranged from 15 to 100 mug depending on the chemical agonist and microvascular bed. Microvascular assessments with acetylcholine revealed a 33-75 % reduction in function following exposure. Additionally, there was a greater sensitivity to CeO2 NP exposure in the mesenteric microvasculature due to the 40 % decrease in the calculated EC50 compared to the coronary microvasculature EC50. CeO2 NP exposure increased mean arterial pressure in some groups. Taken together, these observed microvascular changes may likely have detrimental effects on local blood flow regulation and contribute to cardiovascular dysfunction associated with particle exposure. |
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. |
Distribution and fibrotic response following inhalation exposure to multi-walled carbon nanotubes
Mercer RR , Scabilloni JF , Hubbs AF , Battelli LA , McKinney W , Friend S , Wolfarth MG , Andrew M , Castranova V , Porter DW . Part Fibre Toxicol 2013 10 (1) 33 BACKGROUND: Prior studies have demonstrated a rapid an progressive acute phase response to bolus aspiration of multi-walled carbon nanotubes (MWCNTs). In this study we sought to test the hypothesis that inhalation exposure to MWCNT produces a fibrotic response and that the response is chronically persistent. To address the hypothesis that inhaled MWCNTs cause persistent morphologic changes, male C57BL/6 J mice were exposed in a whole-body inhalation system to a MWCNT aerosol and the fibrotic response in the alveolar region examined at up to 336 days after termination of exposure. METHODS: Inhalation exposure was to a 5 mcg/m3 MWCNT aerosol for 5 hours/day for 12 days (4 times/week for 3 weeks). At the end of inhalation exposures, lungs were either lavaged for analysis of bronchoalveolar lavage (BAL) or preserved by vascular perfusion of fixative while inflated with air at 1, 14, 84, 168 and 336 days post inhalation exposure. Separate, clean-air control groups were also studied. Light microscopy, enhanced darkfield microscopy and field emission electron microscopy (FESEM) of tissue sections were used to analyze the distribution of lung burden following inhalation exposure. Morphometric measurements of Sirius Red staining for fibrillar collagen were used to assess the connective tissue response. Serial section analysis of enhanced darkfield microscope images was used to examine the redistribution of MWCNT fibers within the lungs during the post-exposure period. RESULTS: At day 1 post-exposure 84 +/- 3 and 16 +/- 2 percent of the lung burden (Mean +/- S.E., N = 5) were in the alveolar and airway regions, respectively. Initial distribution within the alveolar region was 56 +/- 5, 7 +/- 4 and 20 +/- 3 percent of lung burden in alveolar macrophages, alveolar airspaces and alveolar tissue, respectively. Clearance reduced the alveolar macrophage burden of MWCNTs by 35 percent between 1 and 168 days post-exposure, while the content of MWCNTs in the alveolar tissue increased by 63 percent. Large MWCNT structures containing greater than 4 fibers were 53.6 percent of the initial lung burden and accounted for the majority of the decline with clearance, while lung burden of singlet MWCNT was essentially unchanged. The mean linear intercept of alveolar airspace, a measure of the expansion of the lungs, was not significantly different between groups. Pulmonary inflammation and damage, measured as the number of polymorphnuclear leukocytes (PMNs) or lactate dehydrogenase activity (LDH) and albumin in BAL, increased rapidly (1 day post) after inhalation of MWCNTs and declined slowly with time post-exposure. The fibrillar collagen in the alveolar region of MWCNT-exposed mice demonstrated a progressive increase in thickness over time (0.17 +/- 0.02, 0.22 +/-0.02, 0.26 +/- 0.03, 0.25 +/- 0.02 and 0.29 +/- 0.01 microns for 1, 14, 84, 168 and 336 days post-exposure) and was significantly different from clean-air controls (0.16 +/- 0.02) at 84 and (0.15 +/- 0.02) at 336 days post-exposure. CONCLUSIONS: Despite the relatively low fraction of the lung burden being delivered to the alveolar tissue, the average thickness of connective tissue in the alveolar region increased by 70% in the 336 days after inhalation exposure. These results demonstrate that inhaled MWCNTs deposit and are retained within the alveolar tissue where they produce a progressive and persistent fibrotic response up to 336 days post-exposure. |
System-based identification of toxicity pathways associated with multi-walled carbon nanotube-induced pathological responses
Snyder-Talkington BN , Dymacek J , Porter DW , Wolfarth MG , Mercer RR , Pacurari M , Denvir J , Castranova V , Qian Y , Guo NL . Toxicol Appl Pharmacol 2013 272 (2) 476-89 The fibrous shape and biopersistence of multi-walled carbon nanotubes (MWCNT) have raised concern over their potential toxicity after pulmonary exposure. As in vivo exposure to MWCNT produced a transient inflammatory and progressive fibrotic response, this study sought to identify significant biological processes associated with lung inflammation and fibrosis pathology data, based upon whole genome mRNA expression, bronchoaveolar lavage scores, and morphometric analysis from C57BL/6J mice exposed by pharyngeal aspiration to 0, 10, 20, 40, or 80mug MWCNT at 1, 7, 28, or 56days post-exposure. Using a novel computational model employing non-negative matrix factorization and Monte Carlo Markov Chain simulation, significant biological processes with expression similar to MWCNT-induced lung inflammation and fibrosis pathology data in mice were identified. A subset of genes in these processes was determined to be functionally related to either fibrosis or inflammation by Ingenuity Pathway Analysis and was used to determine potential significant signaling cascades. Two genes determined to be functionally related to inflammation and fibrosis, vascular endothelial growth factor A (vegfa) and C-C motif chemokine 2 (ccl2), were confirmed by in vitro studies of mRNA and protein expression in small airway epithelial cells exposed to MWCNT as concordant with in vivo expression. This study identified that the novel computational model was sufficient to determine biological processes strongly associated with the pathology of lung inflammation and fibrosis and could identify potential toxicity signaling pathways and mechanisms of MWCNT exposure which could be used for future animal studies to support human risk assessment and intervention efforts. |
Adjuvant effect of zymosan after pulmonary treatment in a mouse ovalbumin allergy model
Young SH , Wolfarth MG , Roberts JR , Kashon ML , Antonini JM . Exp Lung Res 2013 39 (1) 48-57 An association has been observed between indoor mold contamination and lung allergy and asthma. This relationship is not fully understood. 1-->3-beta-Glucan is the major cell wall component of fungi and a good marker of fungi exposure. The objective was to evaluate the adjuvant effect of zymosan, a crude yeast cell wall preparation of 1-->3-beta-glucan, during ovalbumin (OVA) sensitization in an allergy model. BALB/c mice were sensitized by pharyngeal aspiration with saline, 50 mcg of OVA, or OVA with 1, 10, 50, or 75 mcg of zymosan on days 0, 7, and 14. One week after sensitization, each sensitized animal group was challenged with an aspiration dose of 50 mcg of OVA once a week for 2 weeks. At 1 day after the last aspiration, bronchoalveolar lavage fluid and blood was collected, and markers of lung allergy and inflammation were assessed. An adjuvant effect of zymosan on OVA allergy during sensitization was observed as indicated by significant elevations in lung eosinophils, serum OVA-specific IgE, and lung IL-5 in the groups sensitized with zymosan and OVA. Pulmonary treatment with zymosan also amplified lung inflammation. Elevations were observed in lung neutrophils, TNF-alpha, and parameters of lung injury in the groups primed with both zymosan and OVA. In nearly all parameters, a non-linear dose-response relationship was observed in the groups primed with OVA and zymosan. The optimum adjuvant dose of zymosan was 10 mcg. This study demonstrated an adjuvant effect of zymosan when exposures occurred during the sensitization phase in an OVA-induced allergy model in BALB/c mice. |
Multiwalled carbon nanotube-induced gene signatures in the mouse lung: potential predictive value for human lung cancer risk and prognosis.
Guo NL , Wan YW , Denvir J , Porter DW , Pacurari M , Wolfarth MG , Castranova V , Qian Y . J Toxicol Environ Health A 2012 75 (18) 1129-53 Concerns over the potential for multiwalled carbon nanotubes (MWCNT) to induce lung carcinogenesis have emerged. This study sought to (1) identify gene expression signatures in the mouse lungs following pharyngeal aspiration of well-dispersed MWCNT and (2) determine if these genes were associated with human lung cancer risk and progression. Genome-wide mRNA expression profiles were analyzed in mouse lungs (n = 160) exposed to 0, 10, 20, 40, or 80 mcg of MWCNT by pharyngeal aspiration at 1, 7, 28, and 56 d postexposure. By using pairwise statistical analysis of microarray (SAM) and linear modeling, 24 genes were selected, which have significant changes in at least two time points, have a more than 1.5-fold change at all doses, and are significant in the linear model for the dose or the interaction of time and dose. Additionally, a 38-gene set was identified as related to cancer from 330 genes differentially expressed at d 56 postexposure in functional pathway analysis. Using the expression profiles of the cancer-related gene set in 8 mice at d 56 postexposure to 10 mcg of MWCNT, a nearest centroid classification accurately predicts human lung cancer survival with a significant hazard ratio in training set (n = 256) and test set (n = 186). Furthermore, both gene signatures were associated with human lung cancer risk (n = 164) with significant odds ratios. These results may lead to development of a surveillance approach for early detection of lung cancer and prognosis associated with MWCNT in the workplace. |
Differential mouse pulmonary dose- and time course-responses to titanium dioxide nanospheres and nanobelts
Porter DW , Wu N , Hubbs A , Mercer R , Funk K , Meng F , Li J , Wolfarth M , Battelli L , Friend S , Andrew M , Hamilton R , Sriram K , Yang F , Castranova V , Holian A . Toxicol Sci 2012 131 (1) 179-93 Three anatase titanium dioxide (TiO(2)) nanoparticles were prepared; nanospheres (NS), short nanobelts (NB1) and long nanobelts (NB2). These nanoparticles were used to investigate the effect of nanoparticle shape and length on lung toxicity. Mice were exposed (0-30 microg per mouse) by pharyngeal aspiration and pulmonary toxicity was assessed over a 112 day time course. Whole lung lavage data indicated that NB1- and NB2-exposed mice, but not NS-exposed mice, had significant dose- and time-dependent pulmonary inflammation and damage. Histopathological analyses at 112 days post-exposure determined no interstitial fibrosis in any NS-exposed mice, an increased incidence in 30 microg NB1-exposed mice, and significant interstitial fibrosis in 30 microg NB2-exposed mice. At 112 days post-exposure, lung burden of NS was decreased by 96.4% and NB2 by 80.5% from initial deposition levels. At 112 days post-exposure, enhanced dark field microscopy determined that alveolar macrophages were the dominant deposition site, but a fraction of NB1 and NB2 was observed in the alveolar interstitial spaces. For the 30 microg exposure groups at 112 days post-exposure, confocal microscopy and immunofluorescent staining demonstrated that retained NB2 but not NS were present in the interstitium subjacent to the terminal bronchiole near the normal location of the smallest lymphatic capillaries in the lung. These lymphatic capillaries play a critical role in particle clearance, and the accumulation of NB2, but not NS, suggests possible impaired lymphatic clearance by the high aspect ratio particles. In summary, our data indicate that TiO(2) nanoparticle shape alters pulmonary responses, with severity of responses being ranked as NS<NB1<NB2. |
Acute pulmonary dose-responses to inhaled multi-walled carbon nanotubes
Porter DW , Hubbs AF , Chen TB , McKinney W , Mercer RR , Wolfarth MG , Battelli L , Wu N , Sriram K , Leonard S , Andrew ME , Willard P , Sujhi T , Morinobu E , Tsuruoka S , Munekane F , Frazier DG , Castranova V . Nanotoxicology 2012 7 (7) 1179-94 This study investigated the in vivopulmonary toxicity of inhaled multi-walled carbon nanotubes (MWCNT). Mice inhaled aerosolized MWCNT (10 mg/m(3), 5 hours/day) for 2, 4, 8 or 12 days. MWCNT lung burden was linearly related to exposure duration. MWCNT-induced pulmonary inflammation was assessed by determining whole lung lavage (WLL) polymorphonuclear leukocytes (PMN). Lung cytotoxicity was assessed by WLL fluid LDH activities. WLL fluid albumin concentrations were determined as a marker of alveolar air-blood barrier integrity. These parameters significantly increased in MWCNT-exposed mice versus controlsand were dose-dependent. Histopathologic alterations identified in the lung included 1) bronciolocentricinflammation, 2) bronchiolar epithelial hyperplasia and hypertrophy, 3) fibrosis, 4) vascular changes and 5) rare pleural penetration. MWCNT translocated to the lymph node where the deep paracortex was expanded after 8 or 12 days. Acute inhalation of MWCNT induced dosedependent pulmonary inflammation and damage with rapid development of pulmonary fibrosis, and also demonstrated that MWCNT can reach the pleura after inhalation exposure. |
Multi-walled carbon nanotube-induced gene expression in the mouse lung: association with lung pathology.
Pacurari M , Qian Y , Porter DW , Wolfarth M , Wan Y , Luo D , Ding M , Castranova V , Guo NL . Toxicol Appl Pharmacol 2011 255 (1) 18-31 Due to the fibrous shape and durability of multi-walled carbon nanotubes (MWCNT), concerns regarding their potential for producing environmental and human health risks, including carcinogenesis, have been raised. This study sought to investigate how previously identified lung cancer prognostic biomarkers and the related cancer signaling pathways are affected in the mouse lung following pharyngeal aspiration of well-dispersed MWCNT. A total of 63 identified lung cancer prognostic biomarker genes and major signaling biomarker genes were analyzed in mouse lungs (n=80) exposed to 0, 10, 20, 40, or 80mug of MWCNT by pharyngeal aspiration at 7 and 56days post-exposure using quantitative PCR assays. At 7 and 56days post-exposure, a set of 7 genes and a set of 11 genes, respectively, showed differential expression in the lungs of mice exposed to MWCNT vs. the control group. Additionally, these significant genes could separate the control group from the treated group over the time series in a hierarchical gene clustering analysis. Furthermore, 4 genes from these two sets of significant genes, coiled-coil domain containing-99 (Ccdc99), muscle segment homeobox gene-2 (Msx2), nitric oxide synthase-2 (Nos2), and wingless-type inhibitory factor-1 (Wif1), showed significant mRNA expression perturbations at both time points. It was also found that the expression changes of these 4 overlapping genes at 7days post-exposure were attenuated at 56days post-exposure. Ingenuity Pathway Analysis (IPA) found that several carcinogenic-related signaling pathways and carcinogenesis itself were associated with both the 7 and 11 gene signatures. Taken together, this study identifies that MWCNT exposure affects a subset of lung cancer biomarkers in mouse lungs. |
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