BACKGROUND: The unique physicochemical properties of multi-walled carbon nanotubes (MWCNT) have led to many industrial applications. Due to their low density and small size, MWCNT are easily aerosolized in the workplace making respiratory exposures likely in workers. The International Agency for Research on Cancer designated the pristine Mitsui-7 MWCNT (MWCNT-7) as a Group 2B carcinogen, but there was insufficient data to classify all other MWCNT. Previously, MWCNT exposed to high temperature (MWCNT-HT) or synthesized with nitrogen (MWCNT-ND) have been found to elicit attenuated toxicity; however, their genotoxic and carcinogenic potential are not known. Our aim was to measure the genotoxicity of MWCNT-7 compared to these two physicochemically-altered MWCNTs in human lung epithelial cells (BEAS-2B & SAEC). RESULTS: Dose-dependent partitioning of individual nanotubes in the cell nuclei was observed for each MWCNT material and was greatest for MWCNT-7. Exposure to each MWCNT led to significantly increased mitotic aberrations with multi- and monopolar spindle morphologies and fragmented centrosomes. Quantitative analysis of the spindle pole demonstrated significantly increased centrosome fragmentation from 0.024-2.4 mug/mL of each MWCNT. Significant aneuploidy was measured in a dose-response from each MWCNT-7, HT, and ND; the highest dose of 24 mug/mL produced 67, 61, and 55%, respectively. Chromosome analysis demonstrated significantly increased centromere fragmentation and translocations from each MWCNT at each dose. Following 24 h of exposure to MWCNT-7, ND and/or HT in BEAS-2B a significant arrest in the G1/S phase in the cell cycle occurred, whereas the MWCNT-ND also induced a G2 arrest. Primary SAEC exposed for 24 h to each MWCNT elicited a significantly greater arrest in the G1 and G2 phases. However, SAEC arrested in the G1/S phase after 72 h of exposure. Lastly, a significant increase in clonal growth was observed one month after exposure to 0.024 mug/mL MWCNT-HT & ND. CONCLUSIONS: Although MWCNT-HT & ND cause a lower incidence of genotoxicity, all three MWCNTs cause the same type of mitotic and chromosomal disruptions. Chromosomal fragmentation and translocations have not been observed with other nanomaterials. Because in vitro genotoxicity is correlated with in vivo genotoxic response, these studies in primary human lung cells may predict the genotoxic potency in exposed human populations.

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.

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.

Carbon nanotubes are commercially-important products of nanotechnology; however, their low density and small size makes carbon nanotube respiratory exposures likely during their production or processing. We have previously shown mitotic spindle aberrations in cultured primary and immortalized human airway epithelial cells exposed to single-walled carbon nanotubes (SWCNT). In this study, we examined whether multi-walled carbon nanotubes (MWCNT) cause mitotic spindle damage in cultured cells at doses equivalent to 34 years of exposure at the NIOSH Recommended Exposure Limit (REL). MWCNT induced a dose responsive increase in disrupted centrosomes, abnormal mitotic spindles and aneuploid chromosome number 24 hours after exposure to 0.024, 0.24, 2.4 and 24 mug/cm(2) MWCNT. Monopolar mitotic spindles comprised 95% of disrupted mitoses. Three-dimensional reconstructions of 0.1 mum optical sections showed carbon nanotubes integrated with microtubules, DNA and within the centrosome structure. Cell cycle analysis demonstrated a greater number of cells in S-phase and fewer cells in the G2 phase in MWCNT-treated compared to diluent control, indicating a G1/S block in the cell cycle. The monopolar phenotype of the disrupted mitotic spindles and the G1/S block in the cell cycle is in sharp contrast to the multi-polar spindle and G2 block in the cell cycle previously observed following exposure to SWCNT. One month following exposure to MWCNT there was a dramatic increase in both size and number of colonies compared to diluent control cultures, indicating a potential to pass the genetic damage to daughter cells. Our results demonstrate significant disruption of the mitotic spindle by MWCNT at occupationally relevant exposure levels.

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.

Exposure to carbon nanotubes induces significant changes in cellular biomechanics. Using nanoindentation, it is observed that the exposed cells have significantly higher stiffness when compared to controls, especially at the nuclear region, and significant increases in surface area.

Nanotechnology involves technology, science, and engineering in dimensions less than 100 nm. A virtually infinite number of potential nanoscale products can be produced from many different molecules and their combinations. The exponentially increasing number of nanoscale products will solve critical needs in engineering, science, and medicine. However, the virtually infinite number of potential nanotechnology products is a challenge for toxicologic pathologists. Because of their size, nanoparticulates can have therapeutic and toxic effects distinct from micron-sized particulates of the same composition. In the nanoscale, distinct intercellular and intracellular translocation pathways may provide a different distribution than that obtained by micron-sized particulates. Nanoparticulates interact with subcellular structures including microtubules, actin filaments, centrosomes, and chromatin; interactions that may be facilitated in the nanoscale. Features that distinguish nanoparticulates from fine particulates include increased surface area per unit mass and quantum effects. In addition, some nanotechnology products, including the fullerenes, have a novel and reactive surface. Augmented microscopic procedures including enhanced dark-field imaging, immunofluorescence, field-emission scanning electron microscopy, transmission electron microscopy, and confocal microscopy are useful when evaluating nanoparticulate toxicologic pathology. Thus, the pathology assessment is facilitated by understanding the unique features at the nanoscale and the tools that can assist in evaluating nanotoxicology studies.

Carbon nanotubes were among the earliest products of nanotechnology and have many potential applications in medicine, electronics, and manufacturing. The low density, small size, and biological persistence of carbon nanotubes create challenges for exposure control and monitoring and make respiratory exposures to workers likely. We have previously shown mitotic spindle aberrations in cultured primary and immortalized human airway epithelial cells exposed to 24, 48 and 96mcg/cm(2) single-walled carbon nanotubes (SWCNT). To investigate mitotic spindle aberrations at concentrations anticipated in exposed workers, primary and immortalized human airway epithelial cells were exposed to SWCNT for 24-72h at doses equivalent to 20 weeks of exposure at the Permissible Exposure Limit for particulates not otherwise regulated. We have now demonstrated fragmented centrosomes, disrupted mitotic spindles and aneuploid chromosome number at those doses. The data further demonstrated multipolar mitotic spindles comprised 95% of the disrupted mitoses. The increased multipolar mitotic spindles were associated with an increased number of cells in the G2 phase of mitosis, indicating a mitotic checkpoint response. Nanotubes were observed in association with mitotic spindle microtubules, the centrosomes and condensed chromatin in cells exposed to 0.024, 0.24, 2.4 and 24mcg/cm(2) SWCNT. Three-dimensional reconstructions showed carbon nanotubes within the centrosome structure. The lower doses did not cause cytotoxicity or reduction in colony formation after 24h; however, after three days, significant cytotoxicity was observed in the SWCNT-exposed cells. Colony formation assays showed an increased proliferation seven days after exposure. Our results show significant disruption of the mitotic spindle by SWCNT at occupationally relevant doses. The increased proliferation that was observed in carbon nanotube-exposed cells indicates a greater potential to pass the genetic damage to daughter cells. Disruption of the centrosome is common in many solid tumors including lung cancer. The resulting aneuploidy is an early event in the progression of many cancers, suggesting that it may play a role in both tumorigenesis and tumor progression. These results suggest caution should be used in the handling and processing of carbon nanotubes.

Welding generates complex metal aerosols, inhalation of which is linked to adverse health effects among welders. An important health concern of welding fume (WF) exposure is neurological dysfunction akin to Parkinson's disease (PD), thought to be mediated by manganese (Mn) in the fumes. Also, there is a proposition that welding might accelerate the onset of PD. Our recent findings link the presence of Mn in the WF with dopaminergic neurotoxicity seen in rats exposed to manual metal arc-hard surfacing (MMA-HS) or gas metal arc-mild steel (GMA-MS) fumes. To elucidate the molecular mechanisms further, we investigated the association of PD-linked (Park) genes and mitochondrial function in causing dopaminergic abnormality. Repeated instillations of the two fumes at doses that mimic approximately 1 to 5 yr of worker exposure resulted in selective brain accumulation of Mn. This accumulation caused impairment of mitochondrial function and loss of tyrosine hydroxylase (TH) protein, indicative of dopaminergic injury. A fascinating finding was the altered expression of Parkin (Park2), Uchl1 (Park5), and Dj1 (Park7) proteins in dopaminergic brain areas. A similar regimen of manganese chloride (MnCl2) also caused extensive loss of striatal TH, mitochondrial electron transport components, and Park proteins. As mutations in PARK genes have been linked to early-onset PD in humans, and because welding is implicated as a risk factor for parkinsonism, PARK genes might play a critical role in WF-mediated dopaminergic dysfunction. Whether these molecular alterations culminate in neurobehavioral and neuropathological deficits reminiscent of PD remains to be ascertained.

The development of novel engineered nano-sized materials is a rapidly emerging technology with many applications in medicine and industry. In vitro and in vivo studies have suggested many deleterious effects of carbon nanotube exposure including granulomatous inflammation, release of cytosolic enzymes, pulmonary fibrosis, reactive oxygen damage, cellular atypia, DNA fragmentation, mutation and errors in chromosome number as well as mitotic spindle disruption. The physical properties of the carbon nanotubes make respiratory exposure to workers likely during the production or use of commercial products. Many of the investigations of the genotoxicity of carbon nanotubes have focused on reactive oxygen mediated DNA damage; however, the long thin tubular-shaped carbon nanotubes have a striking similarity to cellular microtubules. The similarity of carbon nanotubes to microtubules suggests a potential to interact with cellular biomolecules, such as the mitotic spindle, as well as the motor proteins that separate the chromosomes during cell division. Disruption of centrosomes and mitotic spindles would result in monopolar, tripolar, and quadrapolar divisions of chromosomes. The resulting aneuploidy is a key mechanism in the potential carcinogenicity of carbon nanotubes.

Crystalline silica (silica), a suspected human carcinogen, produces an increase in reactive oxygen species (ROS) when fractured using mechanical tools used in several occupations. Although ROS has been linked to apoptosis, DNA damage, and carcinogenesis, the role of enhanced ROS production by silica in silica-induced carcinogenesis is not completely understood. The goal of this study was to compare freshly fractured and aged silica-induced molecular alterations in human immortalized/transformed bronchial epithelial cells (BEAS-IIB) and lung cancer cells with altered (H460) or deficient (H1299) p53 expression. Exposure to freshly fractured or aged silica produced divergent cellular responses in certain downstream cellular events, including ROS production, apoptosis, cell cycle and chromosomal changes, and gene expression. ROS production increased significantly following exposure to freshly fractured silica compared to aged silica in BEAS-IIB and H460 cells. Apoptosis showed a comparable enhanced level of induction with freshly fractured or aged silica in both cancer lines with p53 functional changes. p53 protein was present in the BEAS-IIB and was absent in cancer cell lines after silica exposure. Exposure to freshly fractured silica also resulted in a rise in aneuploidy in cancer cells with a significantly greater increase in p53-deficient cells. Cytogenetic analysis demonstrated increased metaphase spreads, chromosome breakage, rearrangements, and endoreduplication in both cancer cells. These results suggest that altered and deficient p53 affects the cellular response to freshly fractured silica exposure, and thereby enhances susceptibility and augments cell proliferation and lung cancer development.

Engineered carbon nanotubes are newly emerging manufactured particles with potential applications in electronics, computers, aerospace, and medicine. The low density and small size of these biologically persistent particles makes respiratory exposures to workers likely during the production or use of commercial products. The narrow diameter and great length of single-walled carbon nanotubes (SWCNT) suggest the potential to interact with critical biological structures. To examine the potential of nanotubes to induce genetic damage in normal lung cells, cultured primary and immortalized human airway epithelial cells were exposed to SWCNT or a positive control, vanadium pentoxide. After 24 hr of exposure to either SWCNT or vanadium pentoxide, fragmented centrosomes, multiple mitotic spindle poles, anaphase bridges, and aneuploid chromosome number were observed. Confocal microscopy demonstrated nanotubes within the nucleus that were in association with cellular and mitotic tubulin as well as the chromatin. Our results are the first to report disruption of the mitotic spindle by SWCNT. The nanotube bundles are similar to the size of microtubules that form the mitotic spindle and may be incorporated into the mitotic spindle apparatus. Environ. Mol. Mutagen., 2009. Published 2009 Wiley-Liss, Inc.

Proteasome inhibitors (PIs) are promising new therapeutic agents for treating non-small cell lung carcinoma (NSCLC). To investigate the mechanisms of action of PIs, we analyzed the proapoptotic activities of PIs (MG132 or Bortezomib) in NSCLC cells. We found that both MG132 (>1 muM) and Bortezomib (>0.025 muM) induced a significant apoptosis in NCI-H1703, a PI-sensitive NSCLC cell line, through initially activating the intrinsic apoptosis pathway, leading to the activation of a positive feedback mechanism (PFM), which then conveyed apoptosis signaling from the intrinsic pathway to the extrinsic pathway with formation of a signaling loop for maximal caspase activation. Mcl-1 and Noxa were identified to be the major anti-apoptotic and proapoptotic proteins, respectively, in PI-induced apoptosis and mutually exclusive in protein stability. Although the Mcl-1 protein was upregulated by proteasome inhibition, it was also subjected to caspase 3-dependent cleavage governed by the PFM. Moreover, it was revealed that Mcl-1 protein cleavage contributed to PFM-governed apoptosis in following inter-related ways: reducing the anti-apoptotic Mcl-1; generating the truncated proapoptotic Mcl-1(S); and inducing a shift of balance between Mcl-1 and Noxa. It was further manifested that tumor necrosis factor-related apoptosis-inducing ligand boosted MG132's proapoptotic activity through strengthening the PFM in both NCI-H1703 and NCI-H358, a PI-resistant NSCLC cell line. Therefore, this study provides a basis for enhancing the efficacy of PIs in treating NSCLC.Oncogene advance online publication, 17 August 2009; doi:10.1038/onc.2009.240.