Raymond F. Hamilton

Particle length-dependent titanium dioxide nanomaterials toxicity and bioactivity

BackgroundTitanium dioxide (TiO2) nanomaterials have considerable beneficial uses as photocatalysts and solar cells. It has been established for many years that pigment-grade TiO2 (200 nm sphere) is relatively inert when internalized into a biological model system (in vivo or in vitro). For this reason, TiO2 nanomaterials are considered an attractive alternative in applications where biological exposures will occur. Unfortunately, metal oxides on the nanoscale (one dimension < 100 nm) may or may not exhibit the same toxic potential as the original material. A further complicating issue is the effect of modifying or engineering of the nanomaterial to be structurally and geometrically different from the original material.ResultsTiO2 nanospheres, short (< 5 μm) and long (> 15 μm) nanobelts were synthesized, characterized and tested for biological activity using primary murine alveolar macrophages and in vivo in mice. This study demonstrates that alteration of anatase TiO2 nanomaterial into a fibre structure of greater than 15 μm creates a highly toxic particle and initiates an inflammatory response by alveolar macrophages. These fibre-shaped nanomaterials induced inflammasome activation and release of inflammatory cytokines through a cathepsin B-mediated mechanism. Consequently, long TiO2 nanobelts interact with lung macrophages in a manner very similar to asbestos or silica.ConclusionsThese observations suggest that any modification of a nanomaterial, resulting in a wire, fibre, belt or tube, be tested for pathogenic potential. As this study demonstrates, toxicity and pathogenic potential change dramatically as the shape of the material is altered into one that a phagocytic cell has difficulty processing, resulting in lysosomal disruption.

Silica binding and toxicity in alveolar macrophages.

Inhalation of the crystalline form of silica is associated with a variety of pathologies, from acute lung inflammation to silicosis, in addition to autoimmune disorders and cancer. Basic science investigators looking at the mechanisms involved with the earliest initiators of disease are focused on how the alveolar macrophage interacts with the inhaled silica particle and the consequences of silica-induced toxicity on the cellular level. Based on experimental results, several rationales have been developed for exactly how crystalline silica particles are toxic to the macrophage cell that is functionally responsible for clearance of the foreign particle. For example, silica is capable of producing reactive oxygen species (ROS) either directly (on the particle surface) or indirectly (produced by the cell as a response to silica), triggering cell-signaling pathways initiating cytokine release and apoptosis. With murine macrophages, reactive nitrogen species are produced in the initial respiratory burst in addition to ROS. An alternative explanation for silica toxicity includes lysosomal permeability, by which silica disrupts the normal internalization process leading to cytokine release and cell death. Still other research has focused on the cell surface receptors (collectively known as scavenger receptors) involved in silica binding and internalization. The silica-induced cytokine release and apoptosis are described as the function of receptor-mediated signaling rather than free radical damage. Current research ideas on silica toxicity and binding in the alveolar macrophage are reviewed and discussed.

Interlaboratory evaluation of in vitro cytotoxicity and inflammatory responses to engineered nanomaterials: the NIEHS Nano GO Consortium.

Background: Differences in interlaboratory research protocols contribute to the conflicting data in the literature regarding engineered nanomaterial (ENM) bioactivity. Objectives: Grantees of a National Institute of Health Sciences (NIEHS)-funded consortium program performed two phases of in vitro testing with selected ENMs in an effort to identify and minimize sources of variability. Methods: Consortium program participants (CPPs) conducted ENM bioactivity evaluations on zinc oxide (ZnO), three forms of titanium dioxide (TiO2), and three forms of multiwalled carbon nanotubes (MWCNTs). In addition, CPPs performed bioassays using three mammalian cell lines (BEAS-2B, RLE-6TN, and THP-1) selected in order to cover two different species (rat and human), two different lung epithelial cells (alveolar type II and bronchial epithelial cells), and two different cell types (epithelial cells and macrophages). CPPs also measured cytotoxicity in all cell types while measuring inflammasome activation [interleukin-1β (IL-1β) release] using only THP-1 cells. Results: The overall in vitro toxicity profiles of ENM were as follows: ZnO was cytotoxic to all cell types at ≥ 50 μg/mL, but did not induce IL-1β. TiO2 was not cytotoxic except for the nanobelt form, which was cytotoxic and induced significant IL-1β production in THP-1 cells. MWCNTs did not produce cytotoxicity, but stimulated lower levels of IL-1β production in THP-1 cells, with the original MWCNT producing the most IL-1β. Conclusions: The results provide justification for the inclusion of mechanism-linked bioactivity assays along with traditional cytotoxicity assays for in vitro screening. In addition, the results suggest that conducting studies with multiple relevant cell types to avoid false-negative outcomes is critical for accurate evaluation of ENM bioactivity.

MARCO mediates silica uptake and toxicity in alveolar macrophages from C57Bl/6 mice:

Scavenger receptors (SR), on the surface of the macrophage, appear to be responsible for silica uptake and cell death signaling in the macrophage. The purpose of this study was to isolate which SRs (macrophage receptor with collagenous structure (MARCO), CD204, or CD36) were involved using a variety of SR single and double null mice. The findings indicated that MARCO was the critical SR involved in silica uptake and cytotoxicity in the primary alveolar macrophages (AM) from C57BL/6 mice, as there was no particle uptake or cell death in the absence of this SR. The level of MARCO expression on AM changed significantly with the absence of other SR, and silica uptake was proportional to cell surface MARCO expression. In addition, silica uptake and cytotoxicity were completely blocked by an anti-mouse MARCO antibody. Transfection of Chinese hamster ovary cells with human MARCO supported these conclusions, as silica particles bound to and initiated apoptosis in the MARCO-transfected cells. Strain differences with regard to SR distribution were also examined. There was a differential expression of these SR on AM from each strain, with MARCO dominant for C57BL/6, CD36 dominant on BALB/c, and all three SR expressed on 129/SvJ mice. Similar to the results with C57BL/6 AM, MARCO was involved with silica-induced cell death in the 129/SvJ strain. In contrast, BALB/c AM used an unidentified mechanism for silica uptake because the SR antibodies failed to block particle internalization. Taken together, these results indicate MARCO is the primary AM receptor interacting with silica, depending on mouse strain and level of constitutive expression.

A comparison of dispersing media for various engineered carbon nanoparticles

BackgroundWith the increased manufacture and use of carbon nanoparticles (CNP) there has been increasing concern about the potential toxicity of fugitive CNP in the workplace and ambient environment. To address this matter a number of investigators have conducted in vitro and in vivo toxicity assessments. However, a variety of different approaches for suspension of these particles (culture media, Tween 80, dimethyl sulfoxide, phosphate-buffered saline, fetal calf serum, and others), and different sources of materials have generated potentially conflicting outcomes. The quality of the dispersion of nanoparticles is very dependent on the medium used to suspend them, and this then will most likely affect the biological outcomes.ResultsIn this work, the distributions of different CNP (sources and types) have been characterized in various media. Furthermore, the outcome of instilling the different agglomerates, or size distributions, was examined in mouse lungs after one and seven days. Our results demonstrated that CNP suspended in serum produced particle suspensions with the fewest large agglomerates, and the most uniform distribution in mouse lungs. In addition, no apparent clearance of instilled CNP took place from lungs even after seven days.ConclusionThis work demonstrates that CNP agglomerates are present in all dispersing vehicles to some degree. The vehicle that contains some protein, lipid or protein/lipid component disperses the CNP best, producing fewer large CNP agglomerates. In contrast, vehicles absent of lipid and protein produce the largest CNP agglomerates. The source of the CNP is also a factor in the degree of particle agglomeration within the same vehicle.

Effect of multi-walled carbon nanotube surface modification on bioactivity in the C57BL/6 mouse model

Abstract 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-1β, 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.

Potential involvement of 4-hydroxynonenal in the response of human lung cells to ozone

Ozone is a photochemically generated pollutant that can cause acute pulmonary inflammation and induce cellular injury and may contribute to the development or exacerbation of chronic lung diseases. Despite much research, the mechanisms of ozone- and oxidant-induced cellular injury are still uncertain. Ozone and secondary free radicals have been reported to cause the formation of aldehydes in biological fluids. One of the most toxic aldehydes formed during oxidant-induced lipid peroxidation is 4-hydroxynonenal (HNE). HNE reacts primarily with Cys, Lys, and His amino acids, altering protein function and forming protein adducts. The purpose of this study was to determine whether HNE could account for the acute effects of ozone on lung cells. Human subjects were exposed to 0.4 parts/million ozone or air for 1 h with exercise (each subject served as his/her own control). Six hours after ozone exposure, cells obtained by airway lavage were examined for apoptotic cell injury, and cells from bronchoalveolar lavage were examined for apoptosis, presence of HNE adducts, and expression of stress proteins. Significant apoptosis was evident in airway lung cells after ozone exposure. Western analysis demonstrated an increase in a 32-kDa HNE protein adduct and a number of stress proteins, viz., 72-kDa heat shock protein and ferritin, in alveolar macrophages (AM) after ozone exposure. All of these effects could be replicated by in vitro exposure of AM to HNE. Consequently, the in vitro results and demonstration of HNE protein adducts after ozone exposure are consistent with a potential role for HNE in the cellular toxic effects of ozone.Ozone is a photochemically generated pollutant that can cause acute pulmonary inflammation and induce cellular injury and may contribute to the development or exacerbation of chronic lung diseases. Despite much research, the mechanisms of ozone- and oxidant-induced cellular injury are still uncertain. Ozone and secondary free radicals have been reported to cause the formation of aldehydes in biological fluids. One of the most toxic aldehydes formed during oxidant-induced lipid peroxidation is 4-hydroxynonenal (HNE). HNE reacts primarily with Cys, Lys, and His amino acids, altering protein function and forming protein adducts. The purpose of this study was to determine whether HNE could account for the acute effects of ozone on lung cells. Human subjects were exposed to 0.4 parts/million ozone or air for 1 h with exercise (each subject served as his/her own control). Six hours after ozone exposure, cells obtained by airway lavage were examined for apoptotic cell injury, and cells from bronchoalveolar lavage were examined for apoptosis, presence of HNE adducts, and expression of stress proteins. Significant apoptosis was evident in airway lung cells after ozone exposure. Western analysis demonstrated an increase in a 32-kDa HNE protein adduct and a number of stress proteins, viz., 72-kDa heat shock protein and ferritin, in alveolar macrophages (AM) after ozone exposure. All of these effects could be replicated by in vitro exposure of AM to HNE. Consequently, the in vitro results and demonstration of HNE protein adducts after ozone exposure are consistent with a potential role for HNE in the cellular toxic effects of ozone.

Alveolar macrophage apoptosis and TNF-α, but not p53, expression correlate with murine response to bleomycin

Apoptosis is considered to be a protective mechanism that limits lung injury. However, apoptosis might contribute to the inflammatory burden present in the injured lung. The exposure of mice to bleomycin (BLM) is a well-established model for the study of lung injury. BLM exposure induces DNA damage and enhances tumor necrosis factor (TNF)-alpha expression in the lung. To evaluate the importance of alveolar macrophage (AM) apoptosis in the pathogenesis of lung injury, we exposed BLM-sensitive (C57BL/6) and BLM-resistant (BALB/c) mice to BLM (120 mg/kg) and studied the induction of apoptosis [by light-microscopy changes (2, 8, 12, 24, 48, and 72 h) and annexin V uptake by flow cytometry (24 h)], the secretion of TNF-alpha (measured by ELISA), and the expression of p53 (by immunoblotting) in AM retrieved from these mice. BLM, but not vehicle, induced apoptosis in AM from both murine strains. The numbers of apoptotic AM were significantly greater (P < 0.001) in C57BL/6 mice (52.9%) compared with BALB/c mice (40.8%) as demonstrated by annexin V uptake. BLM induction of apoptosis in AM was preceded by an increased secretion of TNF-alpha in C57BL/6 but not in BALB/c mice. Furthermore, double TNF-alpha receptor-deficient mice, developed on a C57BL/6 background, demonstrated significantly (P < 0.001) lower numbers of apoptotic AM compared with C57BL/6 and BALB/c mice. BLM also enhanced p53 expression in AM from both murine strains. However, p53-deficient mice developed BLM-induced lung injury, exhibited similar lung cell proliferation (measured as proliferating cell nuclear antigen immunostaining), and accumulated similar amounts of lung hydroxyproline (65 +/- 6.9 microgram/lung) as did C57BL/6 (62 +/- 6.5 microgram/lung) mice. Therefore, AM apoptosis is occurring during BLM-induced lung injury in a manner that correlates with murine strain sensitivity to BLM. Furthermore, TNF-alpha secretion rather than p53 expression contributes to the difference in murine strain response to BLM.tumor necrosis factor; strain susceptibility

Differential binding of inorganic particles to MARCO

Alveolar macrophages (AM) in the lung have been documented to play pivotal roles in inflammation and fibrosis (silicosis) following inhalation of crystalline silica (CSiO(2)). In contrast, exposure to either titanium dioxide (TiO(2)) or amorphous silica (ASiO(2)) is considered relatively benign. The scavenger receptor macrophage receptor with collagenous structure (MARCO), expressed on AM, binds and internalizes environmental particles such as silica and TiO(2). Only CSiO(2) is toxic to AM, while ASiO(2) and TiO(2) are not. We hypothesize that differences in induction of pathology between toxic CSiO(2) and nontoxic particles ASiO(2) and TiO(2) may be related to their differential binding to MARCO. In vitro studies with Chinese hamster ovary (CHO) cells transfected with human MARCO and mutants were conducted to better characterize MARCO-particulate (ASiO(2), CSiO(2), and TiO(2)) interactions. Results with MARCO-transfected CHO cells and MARCO-specific antibody demonstrated that the scavenger receptor cysteine-rich (SRCR) domain of MARCO was required for particle binding for all the tested particles. Only TiO(2) required divalent cations (viz., Ca(+2) and/or Mg(+2)) for binding to MARCO, and results from competitive binding studies supported the notion that TiO(2) and both the silica particles bound to different motifs in SRCR domain of MARCO. The results also suggest that particle shape and/or crystal structure may be the determinants linking particle binding to MARCO and cytotoxicity. Taken together, these results demonstrate that the SRCR domain of MARCO is required for particle binding and that involvement of different regions of SRCR domain may distinguish downstream events following particle binding.

Effect of acrolein on human alveolar macrophage NF-κB activity

Acrolein is an environmental pollutant that is known to suppress respiratory host defense against infections; however, the mechanism of the decrease in host defense is not yet clear. We have previously reported that acrolein inhibited endotoxin-induced cytokine release and induced apoptosis in human alveolar macrophages, suggesting that the inhibition of cytokine release and/or cytotoxicity to alveolar macrophages may, in part, be responsible for acrolein-induced immunosuppression in the lung. Because nuclear factor-κB (NF-κB) is an important transcription factor for a number of cytokine genes and is also an important regulator of apoptosis, the effect of acrolein on NF-κB activity was examined by electrophoresis mobility shift assay. Acrolein caused a dose-dependent inhibition of endotoxin-induced NF-κB activation as well as an inhibition of basal level NF-κB activity. Because IκB is a principal regulator of NF-κB activity in the nucleus, changes in IκB were determined by Western blotting. Acrolein-inhibited IκB phosphorylation leads to an increase in cellular IκB levels preventing NF-κB nuclear translocation and is likely the mechanism of acrolein-induced inhibition of NF-κB activity. The role of basal level NF-κB in acrolein-induced apoptosis was also examined. An NF-κB inhibitor (MG-132) also induced apoptosis in human alveolar macrophages, suggesting that a certain basal level NF-κB activity may be required for macrophage cell survival. Taken together, our results suggest that the acrolein-inhibited endotoxin-induced NF-κB activation decreased the basal level NF-κB activity, which may be responsible for the inhibition of cytokine release and the induction of apoptosis in human alveolar macrophages.