Carbon nanotubes are commercially-important products of nanotechnology; however, their low densit... more 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 µg/cm 2 MWCNT. Monopolar mitotic spindles comprised 95% of disrupted mitoses. Three-dimensional reconstructions of 0.1 µm 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.
Carbon nanotubes (CNTs) exhibit many unique properties that allow their use in a wide spectrum of... more Carbon nanotubes (CNTs) exhibit many unique properties that allow their use in a wide spectrum of biomedical applications, such as delivery of therapeutically molecules, genes and drugs. However, advances in the biomedical applications of CNTs are being hindered by many uncertainties regarding their cellular uptake mechanisms and fate inside the body. Reports have shown that CNTs toxicity can be attributed to their physical and chemical properties. However, there are no compelling reports that provide fundamental understanding of the toxicological and pharmacological profiles of cellular systems exposed to CNTs. In this research, an electronic platform is used to assess the toxicity of CNTs when incubated with human lung epithelial cells in vitro. This non-invasive technique relies on an electrical cell impedance sensing system (ECIS) used as a proxy to assess changes in cellular morphology and cell-cell interactions upon exposure to different concentrations and functionalities of C...
Carbon nanotubes are being explored for broadest applications including electrical circuits, hydr... more Carbon nanotubes are being explored for broadest applications including electrical circuits, hydrogen storage, fiber optics, and conductive plastics. In recent years, their biocompatibility with small molecules, such as proteins and nucleic acids, opened up exciting biomedical applications. Thus, concerns about their potential cytotoxicity on both human and environmental have risen. It is necessary to develop significant and sensitive analysis technologies for carbon nanotubes cytotoxicity investigation. Our research aims at elucidating the mechanisms of interactions of carbon nanotubes with biological systems and evaluating the overall effects on the cell fate. To unravel the molecular mechanisms of such interaction, we need to use the latest technological advances in which both structure and function of the cells upon exposure are being analyzed. Here we employed Atomic Force Microscopy (AFM) to carry on system analysis on cellular topography and mechanical properties of immobiliz...
Topological analysis of cells and subcellular structures is one of the major trends in biology. S... more Topological analysis of cells and subcellular structures is one of the major trends in biology. Since the spatial and temporal changes of the mechanical properties of living cells reflect complex underlying physiological processes, there have been considerable efforts in following these changes and providing valuable insight into the biological importance of cellular mechanics and its regulation. In here we propose to study the cell mechanics and regulation in response to exposure to nanomaterials. Nanomaterials have been recently used in biolabeling, biodetection, biomolecule delivery, bioseparation or regenerative medicine and tissue engineering. Specifically, we use Atomic Force Microscopy (AFM) for probing cellular topography and studying mechanical properties of immobilized and live cells, and cells exposed to nanomaterials. Our aim is to produce robust, internally quantitative maps of relative elasticity of fixed cells and compare those maps to those of live cells, and cells i...
Carbon nanotubes (CNTs) exhibit unique properties that make them attractive candidates for the de... more Carbon nanotubes (CNTs) exhibit unique properties that make them attractive candidates for the delivery of therapeutic molecules, genes and drugs. However, advances in the biomedical applications of CNTs are being hindered by uncertainties associated with their cellular uptake and delivery mechanisms as well as their fate inside the biological systems. Recent reports have shown that CNTs toxicity can be attributed to metal impurities, length, size, coating, uptake, or internalization. However, there are no reports that provide fundamental understanding of the toxicological and pharmacological profiles of cellular systems exposed to CNTs. In this research, a new approach is used to assess the toxicity of a wide library of surface modified CNTs incubated with human lung epithelial cells (BEAS-2B) in real time. This non-invasive technique relies on an electrical cell impedance sensing system (ECIS) used as a proxy to measure morphological changes and cellular interactions upon exposure...
Carbon nanotubes (CNTs) are applied for a variety of applications from nanocircuits, to hydrogen ... more Carbon nanotubes (CNTs) are applied for a variety of applications from nanocircuits, to hydrogen storage devices, and from opticals fibers to conductive plastics. Recently, their functionalization with biomolecules offer opportunities for exciting biological and biomedical applications in drug delivery or bioimaging. However, because of their interactions with biological systems and their ability to translocate and persist into the circulatory and lymphatic systems and tissues, concerns about CNTs intrinsic toxicity have risen. It is thus necessary to develop significant and sensitive advances analysis technologies for real time investigation of CNTs toxicity. Our research aims at elucidating the mechanisms involved in CNTs internalization and association with cellular systems by evaluating the overall effects on of CNTs on cellular biophysical properties-biomechanics. To unravel the biomolecular mechanisms of such interactions, we used Fluorescence Activated Cell Sorting (FACS) and...
Inhalation exposure to multi-walled carbon nanotubes (MWCNT) in mice results in inflammation, fib... more 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 μg g À1 body weight) or vehicle (corn oil). One week after injections, mice were exposed by inhalation to MWCNT (5 mg m À3 , 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. . Schematic of mouse exposure groups and analysis. B. N. Snyder-Talkington et al. /journal/jat * Hyperplasia refers to both focal alveolar and bronchiolo-alveolar hyperplasia. Reasons for having fewer blood samples than the total number of mice in a treatment category included insufficient blood draws, insufficient mRNA or miRNA quantity or quality for analysis, and early death of a mouse that prohibited whole blood collection. MAC, methylcholanthrene; MWCNT, multi-walled carbon nanotubes. B. N. Snyder-Talkington et al.
Cytogenetic changes that occur during the progression of rat hepatocarcinogenesis were assessed w... more Cytogenetic changes that occur during the progression of rat hepatocarcinogenesis were assessed with three rat liver epithelial cell lines derived from WB cells. Previously characterized WBneo, WBras, and WBrasIIa cells were grown in culture and analyzed for structural and numerical chromosomal integrity by banded karyotype analysis. The WBneo cells had a low level of aneuploidy with a consistent loss of the Y chromosome by passage 7. The ras-transfected cell line selected for growth in soft agar, WBras, had acquired a loss of chromosome 3 (12%) or 3p (34%), a trisomy of chromosome 1, as well as the chromosome Y loss. The cell line produced from tumors generated by injection of the WBras cells into a syngeneic F344 rat, WBrasIIa, contained additional chromosomal changes. The WBrasIIa line comprised cells retaining a trisomy of chromosome 1 (55%) and cells with two copies of chromosome 1, with a minimal duplication of 1q3.7 to 1q4.3 (45%). This tumor-derived cell line contained, in a...
Although adenocarcinoma is rapidly becoming the most common form of lung cancer in the United Sta... more Although adenocarcinoma is rapidly becoming the most common form of lung cancer in the United States, the difficulty in obtaining lung cancer families and representative samples of the various stages of adenocarcinoma progression has led to intense study of mouse models. As a powerful approach to delineating molecular changes, we have analyzed 15 early-passage mouse cell lines by spectral karyotyping. Entire copies of chromosomes 1, 2, 6, 12, 15, and 19 were gained, and entire copies of chromosomes 4, 7, 8, and 14 were lost. Significant gains of portions of chromosome 1 (93% of the tumor cell lines analyzed), chromosome 2 (53%), chromosome 6 (73%), chromosome 7 (80%), chromosome 12 (47%), and chromosome 15 (73%) and partial loss of chromosome 4 (87%), chromosome 7 (80%), chromosome 8 (53%), chromosome 10 (33%), and chromosome 14 (33%) were observed. Recurrent translocations included 10:del(10)(A1::C1), t(4;8)(C4;A1), and der (1;12)(C2;C2). The minimal regions of chromosomal alterati...
Engineered carbon nanotubes are currently used in many consumer and industrial products such as p... more 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 μg/g BW, i.p.), or vehicle (corn oil). One week after i.p. injections, mice were exposed ...
Toxicity of engineered nanomaterials is associated with their inherent properties, both physical ... more Toxicity of engineered nanomaterials is associated with their inherent properties, both physical and chemical. Recent studies have shown that exposure to multi-walled carbon nanotubes (MWCNTs) promotes tumors and tumor-associated pathologies and lead to carcinogenesis in model in vivo systems. Here in we examined the potential of purified MWCNTs used at occupationally relevant exposure doses for particles not otherwise regulated to affect human lung epithelial cells. The uptake of the purified MWCNTs was evaluated using fluorescence activated cell sorting (FACS), while the effects on cell fate were assessed using 2- (4-iodophenyl) - 3- (4-nitrophenyl) - 5-(2, 4-disulfophenyl) -2H-tetrazolium salt colorimetric assay, cell cycle and nanoindentation. Our results showed that exposure to MWCNTs reduced cell metabolic activity and induced cell cycle arrest. Our analysis further emphasized that MWCNTs-induced cellular fate results from multiple types of interactions that could be analyzed by means of intracellular biomechanical changes and are pivotal in understanding the underlying MWCNTs-induced cell transformation.
Carbon nanotubes are commercially-important products of nanotechnology; however, their low densit... more 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 µg/cm 2 MWCNT. Monopolar mitotic spindles comprised 95% of disrupted mitoses. Three-dimensional reconstructions of 0.1 µm 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.
The incidence of adenocarcinoma of the lung is increasing in the United States, however, the diff... more The incidence of adenocarcinoma of the lung is increasing in the United States, however, the difficulties in obtaining lung cancer families and representative samples of early to late stages of the disease have lead to the study of mouse models for lung cancer. We used Spectral Karyotyping (SKY), mapping with fluorescently labeled genomic clones (FISH), comparative genomic hybridization (CGH) arrays, gene expression arrays, Western immunoblot and real time polymerase chain reaction (PCR) to analyze nine pairs of high-invasive and low-invasive tumor cell strains derived from early passage mouse lung adenocarcinoma cells to detect molecular changes associated with tumor invasion. The duplication of chromosomes 1 and 15 and deletion of chromosome 8 were significantly associated with a high-invasive phenotype. The duplication of chromosome 1 at band C4 and E1/2-H1 were the most significant chromosomal changes in the high-invasive cell strains. Mapping with FISH and CGH array further narrowed the minimum region of duplication of chromosome 1 to 71-82 centimorgans (cM). Expression array analysis and confirmation by real time PCR demonstrated increased expression of COX-2, Translin (TB-RBP), DYRK3, NUCKS and Tubulin-alpha4 genes in the high-invasive cell strains. Elevated expression and copy number of these genes, which are involved in inflammation, cell movement, proliferation, inhibition of apoptosis and telomere elongation, were associated with an invasive phenotype. Similar linkage groups are altered in invasive human lung adenocarcinoma, implying that the mouse is a valid genetic model for the study of the progression of human lung adenocarcinoma.
The production of carbon nanofibers and nanotubes (CNF/CNT) and their composite products is incre... more The production of carbon nanofibers and nanotubes (CNF/CNT) and their composite products is increasing globally. CNF are generating great interest in industrial sectors such as energy production and electronics, where alternative materials may have limited performance or are produced at a much higher cost. However, despite the increasing industrial use of carbon nanofibers, information on their potential adverse health effects is limited. In the current study, we examine the cytotoxic and genotoxic potential of carbon-based nanofibers (Pyrograf®-III) and compare this material with the effects of asbestos fibers (crocidolite) or singlewalled carbon nanotubes (SWCNT). The genotoxic effects in the lung fibroblast (V79) cell line were examined using two complementary assays: the comet assay and micronucleus (MN) test. In addition, we utilized fluorescence in situ hybridization to detect the chromatin pan-centromeric signals within the MN indicating their origin by aneugenic (chromosomal malsegregation) or clastogenic (chromosome breakage) mechanisms. Cytotoxicity tests revealed a concentration-and time-dependent loss of V79 cell viability after exposure to all tested materials in the following sequence: asbestos N CNF N SWCNT. Additionally, cellular uptake and generation of oxygen radicals was seen in the murine RAW264.7 macrophages following exposure to CNF or asbestos but not after administration of SWCNT. DNA damage and MN induction were found after exposure to all tested materials with the strongest effect seen for CNF. Finally, we demonstrated that CNF induced predominately centromere-positive MN in primary human small airway epithelial cells (SAEC) indicating aneugenic events. Further investigations are warranted to elucidate the possible mechanisms involved in CNF-induced genotoxicity.
Mechanisms of digitoxin-inhibited cell growth and induced apoptosis in human non-small cell lung ... more Mechanisms of digitoxin-inhibited cell growth and induced apoptosis in human non-small cell lung cancer (NCI-H460) cells remain unclear. Understanding how digitoxin or derivate analogs induce their cytotoxic effect below therapeutically relevant concentrations will help in designing and developing novel, safer and more effective anti-cancer drugs. In this study, NCI-H460 cells were treated with digitoxin and a synthetic analog D6-MA to determine their anti-cancer activity. Different concentrations of digitoxin and D6-MA were used and the subsequent changes in cell morphology, viability, cell cycle, and protein expressions were determined. Digitoxin and D6-MA induced dose-dependent apoptotic morphologic changes in NCI-H460 cells via caspase-9 cleavage, with D6-MA possessing 5-fold greater potentcy than digitoxin. In comparison, nontumorigenic immortalized bronchial and small airway epithelial cells displayed significantly less apoptotic sensitivity compared to NCI-H460 cells suggesting that both digitoxin and D6-MA were selective for NSCLC. Furthermore, NCI-H460 cells arrested in G(2)/M phase following digitoxin and D6-MA treatment. Post-treatment evaluation of key G2/M checkpoint regulatory proteins identified down-regulation of cyclin B1/cdc2 complex and survivin. Additionally, Chk1/2 and p53 related proteins experienced down-regulation suggesting a p53-independent cell cycle arrest mechanism. In summary, digitoxin and D6-MA exert anti-cancer effects on NCI-H460 cells through apoptosis or cell cycle arrest, with D6-MA showing at least 5-fold greater potency relative to digitoxin.
Mutation Research/Genetic Toxicology and Environmental Mutagenesis, 2012
Engineered carbon nanotubes are newly emerging manufactured particles with potential applications... more 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 singlewalled carbon nanotubes suggest the potential to interact with critical biological structures.
Six different techniques were evaluated to define better those technical factors that are most cr... more Six different techniques were evaluated to define better those technical factors that are most critical for obtaining prometaphase cells for banding analysis. Our results demonstrate: (a) colcemid exposures of 30 min or less have no effect on increasing the yield of prometaphase cells, (b) colcemid exposures of greater than 0.1 #g/ml can be toxic, (c) methotrexate depresses the mitotic index significantly and seems to increase the incidence of prometaphase cells only because it suppresses later forms; and (d) the optimum number of cytogenetically satisfactory prometaphase cells can be obtained with a 4-h exposure to a combination of low concentration actinomycin D (0.5 ttg/ml) and colcemid (0.1 #g/ml). This technique inhibits chromosome condensation while permitting prometaphase cells to accumulate for 4 h.
Since tamoxifen is efficacious for the prevention of second primary breast neoplasms in humans an... more Since tamoxifen is efficacious for the prevention of second primary breast neoplasms in humans and has a low reported incidence of acute side effects, several structurally related compounds have been developed for the treatment of breast cancer including toremifene and idoxifene. We have compared the karyotypic alterations that occur after a single per os administration of 35 mg/kg of tamoxifen, toremifene or idoxifene to female Sprague-Dawley rats. One day following treatment, the rats were sacrificed and the hepatocytes isolated and cultured. After 47 h in culture, colcemid was added for 3 h prior to harvest of the hepatocytes for karyotypic evaluation. At least 100 metaphase spreads were examined for each of five rats per treatment. Toremifene resulted in aneuploidy in 50±7% of the cells examined and idoxifene induced a 57±4% aneuploidy compared with the 85±7% level induced by tamoxifen. Since the level of aneuploidy in solvent-treated rats was 3±3%, the induction of aneuploidy in at least 50% of the cells from rats treated with tamoxifen, toremifene or idoxifene was highly significant. Analysis of electron micrographs of cultures treated with these antiestrogens demonstrated a range of phenotypes including multipolar spindles in toremifene-treated rats and condensed chromosomes in the presence of an intact nuclear envelope in occasional idoxifene-treated rat hepatocytes. The exclusion of chromosomes from the spindle apparatus and the lagging of some chromosomes on the metaphase plate correlate with the high rate of induction of aneuploidy in the rat liver as determined by karyotypic analysis of hepatocytes from rats treated with these triphenylethylenes.
Carbon nanotubes are commercially-important products of nanotechnology; however, their low densit... more 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 µg/cm 2 MWCNT. Monopolar mitotic spindles comprised 95% of disrupted mitoses. Three-dimensional reconstructions of 0.1 µm 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.
Carbon nanotubes (CNTs) exhibit many unique properties that allow their use in a wide spectrum of... more Carbon nanotubes (CNTs) exhibit many unique properties that allow their use in a wide spectrum of biomedical applications, such as delivery of therapeutically molecules, genes and drugs. However, advances in the biomedical applications of CNTs are being hindered by many uncertainties regarding their cellular uptake mechanisms and fate inside the body. Reports have shown that CNTs toxicity can be attributed to their physical and chemical properties. However, there are no compelling reports that provide fundamental understanding of the toxicological and pharmacological profiles of cellular systems exposed to CNTs. In this research, an electronic platform is used to assess the toxicity of CNTs when incubated with human lung epithelial cells in vitro. This non-invasive technique relies on an electrical cell impedance sensing system (ECIS) used as a proxy to assess changes in cellular morphology and cell-cell interactions upon exposure to different concentrations and functionalities of C...
Carbon nanotubes are being explored for broadest applications including electrical circuits, hydr... more Carbon nanotubes are being explored for broadest applications including electrical circuits, hydrogen storage, fiber optics, and conductive plastics. In recent years, their biocompatibility with small molecules, such as proteins and nucleic acids, opened up exciting biomedical applications. Thus, concerns about their potential cytotoxicity on both human and environmental have risen. It is necessary to develop significant and sensitive analysis technologies for carbon nanotubes cytotoxicity investigation. Our research aims at elucidating the mechanisms of interactions of carbon nanotubes with biological systems and evaluating the overall effects on the cell fate. To unravel the molecular mechanisms of such interaction, we need to use the latest technological advances in which both structure and function of the cells upon exposure are being analyzed. Here we employed Atomic Force Microscopy (AFM) to carry on system analysis on cellular topography and mechanical properties of immobiliz...
Topological analysis of cells and subcellular structures is one of the major trends in biology. S... more Topological analysis of cells and subcellular structures is one of the major trends in biology. Since the spatial and temporal changes of the mechanical properties of living cells reflect complex underlying physiological processes, there have been considerable efforts in following these changes and providing valuable insight into the biological importance of cellular mechanics and its regulation. In here we propose to study the cell mechanics and regulation in response to exposure to nanomaterials. Nanomaterials have been recently used in biolabeling, biodetection, biomolecule delivery, bioseparation or regenerative medicine and tissue engineering. Specifically, we use Atomic Force Microscopy (AFM) for probing cellular topography and studying mechanical properties of immobilized and live cells, and cells exposed to nanomaterials. Our aim is to produce robust, internally quantitative maps of relative elasticity of fixed cells and compare those maps to those of live cells, and cells i...
Carbon nanotubes (CNTs) exhibit unique properties that make them attractive candidates for the de... more Carbon nanotubes (CNTs) exhibit unique properties that make them attractive candidates for the delivery of therapeutic molecules, genes and drugs. However, advances in the biomedical applications of CNTs are being hindered by uncertainties associated with their cellular uptake and delivery mechanisms as well as their fate inside the biological systems. Recent reports have shown that CNTs toxicity can be attributed to metal impurities, length, size, coating, uptake, or internalization. However, there are no reports that provide fundamental understanding of the toxicological and pharmacological profiles of cellular systems exposed to CNTs. In this research, a new approach is used to assess the toxicity of a wide library of surface modified CNTs incubated with human lung epithelial cells (BEAS-2B) in real time. This non-invasive technique relies on an electrical cell impedance sensing system (ECIS) used as a proxy to measure morphological changes and cellular interactions upon exposure...
Carbon nanotubes (CNTs) are applied for a variety of applications from nanocircuits, to hydrogen ... more Carbon nanotubes (CNTs) are applied for a variety of applications from nanocircuits, to hydrogen storage devices, and from opticals fibers to conductive plastics. Recently, their functionalization with biomolecules offer opportunities for exciting biological and biomedical applications in drug delivery or bioimaging. However, because of their interactions with biological systems and their ability to translocate and persist into the circulatory and lymphatic systems and tissues, concerns about CNTs intrinsic toxicity have risen. It is thus necessary to develop significant and sensitive advances analysis technologies for real time investigation of CNTs toxicity. Our research aims at elucidating the mechanisms involved in CNTs internalization and association with cellular systems by evaluating the overall effects on of CNTs on cellular biophysical properties-biomechanics. To unravel the biomolecular mechanisms of such interactions, we used Fluorescence Activated Cell Sorting (FACS) and...
Inhalation exposure to multi-walled carbon nanotubes (MWCNT) in mice results in inflammation, fib... more 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 μg g À1 body weight) or vehicle (corn oil). One week after injections, mice were exposed by inhalation to MWCNT (5 mg m À3 , 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. . Schematic of mouse exposure groups and analysis. B. N. Snyder-Talkington et al. /journal/jat * Hyperplasia refers to both focal alveolar and bronchiolo-alveolar hyperplasia. Reasons for having fewer blood samples than the total number of mice in a treatment category included insufficient blood draws, insufficient mRNA or miRNA quantity or quality for analysis, and early death of a mouse that prohibited whole blood collection. MAC, methylcholanthrene; MWCNT, multi-walled carbon nanotubes. B. N. Snyder-Talkington et al.
Cytogenetic changes that occur during the progression of rat hepatocarcinogenesis were assessed w... more Cytogenetic changes that occur during the progression of rat hepatocarcinogenesis were assessed with three rat liver epithelial cell lines derived from WB cells. Previously characterized WBneo, WBras, and WBrasIIa cells were grown in culture and analyzed for structural and numerical chromosomal integrity by banded karyotype analysis. The WBneo cells had a low level of aneuploidy with a consistent loss of the Y chromosome by passage 7. The ras-transfected cell line selected for growth in soft agar, WBras, had acquired a loss of chromosome 3 (12%) or 3p (34%), a trisomy of chromosome 1, as well as the chromosome Y loss. The cell line produced from tumors generated by injection of the WBras cells into a syngeneic F344 rat, WBrasIIa, contained additional chromosomal changes. The WBrasIIa line comprised cells retaining a trisomy of chromosome 1 (55%) and cells with two copies of chromosome 1, with a minimal duplication of 1q3.7 to 1q4.3 (45%). This tumor-derived cell line contained, in a...
Although adenocarcinoma is rapidly becoming the most common form of lung cancer in the United Sta... more Although adenocarcinoma is rapidly becoming the most common form of lung cancer in the United States, the difficulty in obtaining lung cancer families and representative samples of the various stages of adenocarcinoma progression has led to intense study of mouse models. As a powerful approach to delineating molecular changes, we have analyzed 15 early-passage mouse cell lines by spectral karyotyping. Entire copies of chromosomes 1, 2, 6, 12, 15, and 19 were gained, and entire copies of chromosomes 4, 7, 8, and 14 were lost. Significant gains of portions of chromosome 1 (93% of the tumor cell lines analyzed), chromosome 2 (53%), chromosome 6 (73%), chromosome 7 (80%), chromosome 12 (47%), and chromosome 15 (73%) and partial loss of chromosome 4 (87%), chromosome 7 (80%), chromosome 8 (53%), chromosome 10 (33%), and chromosome 14 (33%) were observed. Recurrent translocations included 10:del(10)(A1::C1), t(4;8)(C4;A1), and der (1;12)(C2;C2). The minimal regions of chromosomal alterati...
Engineered carbon nanotubes are currently used in many consumer and industrial products such as p... more 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 μg/g BW, i.p.), or vehicle (corn oil). One week after i.p. injections, mice were exposed ...
Toxicity of engineered nanomaterials is associated with their inherent properties, both physical ... more Toxicity of engineered nanomaterials is associated with their inherent properties, both physical and chemical. Recent studies have shown that exposure to multi-walled carbon nanotubes (MWCNTs) promotes tumors and tumor-associated pathologies and lead to carcinogenesis in model in vivo systems. Here in we examined the potential of purified MWCNTs used at occupationally relevant exposure doses for particles not otherwise regulated to affect human lung epithelial cells. The uptake of the purified MWCNTs was evaluated using fluorescence activated cell sorting (FACS), while the effects on cell fate were assessed using 2- (4-iodophenyl) - 3- (4-nitrophenyl) - 5-(2, 4-disulfophenyl) -2H-tetrazolium salt colorimetric assay, cell cycle and nanoindentation. Our results showed that exposure to MWCNTs reduced cell metabolic activity and induced cell cycle arrest. Our analysis further emphasized that MWCNTs-induced cellular fate results from multiple types of interactions that could be analyzed by means of intracellular biomechanical changes and are pivotal in understanding the underlying MWCNTs-induced cell transformation.
Carbon nanotubes are commercially-important products of nanotechnology; however, their low densit... more 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 µg/cm 2 MWCNT. Monopolar mitotic spindles comprised 95% of disrupted mitoses. Three-dimensional reconstructions of 0.1 µm 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.
The incidence of adenocarcinoma of the lung is increasing in the United States, however, the diff... more The incidence of adenocarcinoma of the lung is increasing in the United States, however, the difficulties in obtaining lung cancer families and representative samples of early to late stages of the disease have lead to the study of mouse models for lung cancer. We used Spectral Karyotyping (SKY), mapping with fluorescently labeled genomic clones (FISH), comparative genomic hybridization (CGH) arrays, gene expression arrays, Western immunoblot and real time polymerase chain reaction (PCR) to analyze nine pairs of high-invasive and low-invasive tumor cell strains derived from early passage mouse lung adenocarcinoma cells to detect molecular changes associated with tumor invasion. The duplication of chromosomes 1 and 15 and deletion of chromosome 8 were significantly associated with a high-invasive phenotype. The duplication of chromosome 1 at band C4 and E1/2-H1 were the most significant chromosomal changes in the high-invasive cell strains. Mapping with FISH and CGH array further narrowed the minimum region of duplication of chromosome 1 to 71-82 centimorgans (cM). Expression array analysis and confirmation by real time PCR demonstrated increased expression of COX-2, Translin (TB-RBP), DYRK3, NUCKS and Tubulin-alpha4 genes in the high-invasive cell strains. Elevated expression and copy number of these genes, which are involved in inflammation, cell movement, proliferation, inhibition of apoptosis and telomere elongation, were associated with an invasive phenotype. Similar linkage groups are altered in invasive human lung adenocarcinoma, implying that the mouse is a valid genetic model for the study of the progression of human lung adenocarcinoma.
The production of carbon nanofibers and nanotubes (CNF/CNT) and their composite products is incre... more The production of carbon nanofibers and nanotubes (CNF/CNT) and their composite products is increasing globally. CNF are generating great interest in industrial sectors such as energy production and electronics, where alternative materials may have limited performance or are produced at a much higher cost. However, despite the increasing industrial use of carbon nanofibers, information on their potential adverse health effects is limited. In the current study, we examine the cytotoxic and genotoxic potential of carbon-based nanofibers (Pyrograf®-III) and compare this material with the effects of asbestos fibers (crocidolite) or singlewalled carbon nanotubes (SWCNT). The genotoxic effects in the lung fibroblast (V79) cell line were examined using two complementary assays: the comet assay and micronucleus (MN) test. In addition, we utilized fluorescence in situ hybridization to detect the chromatin pan-centromeric signals within the MN indicating their origin by aneugenic (chromosomal malsegregation) or clastogenic (chromosome breakage) mechanisms. Cytotoxicity tests revealed a concentration-and time-dependent loss of V79 cell viability after exposure to all tested materials in the following sequence: asbestos N CNF N SWCNT. Additionally, cellular uptake and generation of oxygen radicals was seen in the murine RAW264.7 macrophages following exposure to CNF or asbestos but not after administration of SWCNT. DNA damage and MN induction were found after exposure to all tested materials with the strongest effect seen for CNF. Finally, we demonstrated that CNF induced predominately centromere-positive MN in primary human small airway epithelial cells (SAEC) indicating aneugenic events. Further investigations are warranted to elucidate the possible mechanisms involved in CNF-induced genotoxicity.
Mechanisms of digitoxin-inhibited cell growth and induced apoptosis in human non-small cell lung ... more Mechanisms of digitoxin-inhibited cell growth and induced apoptosis in human non-small cell lung cancer (NCI-H460) cells remain unclear. Understanding how digitoxin or derivate analogs induce their cytotoxic effect below therapeutically relevant concentrations will help in designing and developing novel, safer and more effective anti-cancer drugs. In this study, NCI-H460 cells were treated with digitoxin and a synthetic analog D6-MA to determine their anti-cancer activity. Different concentrations of digitoxin and D6-MA were used and the subsequent changes in cell morphology, viability, cell cycle, and protein expressions were determined. Digitoxin and D6-MA induced dose-dependent apoptotic morphologic changes in NCI-H460 cells via caspase-9 cleavage, with D6-MA possessing 5-fold greater potentcy than digitoxin. In comparison, nontumorigenic immortalized bronchial and small airway epithelial cells displayed significantly less apoptotic sensitivity compared to NCI-H460 cells suggesting that both digitoxin and D6-MA were selective for NSCLC. Furthermore, NCI-H460 cells arrested in G(2)/M phase following digitoxin and D6-MA treatment. Post-treatment evaluation of key G2/M checkpoint regulatory proteins identified down-regulation of cyclin B1/cdc2 complex and survivin. Additionally, Chk1/2 and p53 related proteins experienced down-regulation suggesting a p53-independent cell cycle arrest mechanism. In summary, digitoxin and D6-MA exert anti-cancer effects on NCI-H460 cells through apoptosis or cell cycle arrest, with D6-MA showing at least 5-fold greater potency relative to digitoxin.
Mutation Research/Genetic Toxicology and Environmental Mutagenesis, 2012
Engineered carbon nanotubes are newly emerging manufactured particles with potential applications... more 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 singlewalled carbon nanotubes suggest the potential to interact with critical biological structures.
Six different techniques were evaluated to define better those technical factors that are most cr... more Six different techniques were evaluated to define better those technical factors that are most critical for obtaining prometaphase cells for banding analysis. Our results demonstrate: (a) colcemid exposures of 30 min or less have no effect on increasing the yield of prometaphase cells, (b) colcemid exposures of greater than 0.1 #g/ml can be toxic, (c) methotrexate depresses the mitotic index significantly and seems to increase the incidence of prometaphase cells only because it suppresses later forms; and (d) the optimum number of cytogenetically satisfactory prometaphase cells can be obtained with a 4-h exposure to a combination of low concentration actinomycin D (0.5 ttg/ml) and colcemid (0.1 #g/ml). This technique inhibits chromosome condensation while permitting prometaphase cells to accumulate for 4 h.
Since tamoxifen is efficacious for the prevention of second primary breast neoplasms in humans an... more Since tamoxifen is efficacious for the prevention of second primary breast neoplasms in humans and has a low reported incidence of acute side effects, several structurally related compounds have been developed for the treatment of breast cancer including toremifene and idoxifene. We have compared the karyotypic alterations that occur after a single per os administration of 35 mg/kg of tamoxifen, toremifene or idoxifene to female Sprague-Dawley rats. One day following treatment, the rats were sacrificed and the hepatocytes isolated and cultured. After 47 h in culture, colcemid was added for 3 h prior to harvest of the hepatocytes for karyotypic evaluation. At least 100 metaphase spreads were examined for each of five rats per treatment. Toremifene resulted in aneuploidy in 50±7% of the cells examined and idoxifene induced a 57±4% aneuploidy compared with the 85±7% level induced by tamoxifen. Since the level of aneuploidy in solvent-treated rats was 3±3%, the induction of aneuploidy in at least 50% of the cells from rats treated with tamoxifen, toremifene or idoxifene was highly significant. Analysis of electron micrographs of cultures treated with these antiestrogens demonstrated a range of phenotypes including multipolar spindles in toremifene-treated rats and condensed chromosomes in the presence of an intact nuclear envelope in occasional idoxifene-treated rat hepatocytes. The exclusion of chromosomes from the spindle apparatus and the lagging of some chromosomes on the metaphase plate correlate with the high rate of induction of aneuploidy in the rat liver as determined by karyotypic analysis of hepatocytes from rats treated with these triphenylethylenes.
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Papers by Linda Sargent