scholarly journals Few-layer graphene induces both primary and secondary genotoxicity in epithelial barrier models in vitro

2021 ◽  
Vol 19 (1) ◽  
Author(s):  
Michael J. Burgum ◽  
Martin J. D. Clift ◽  
Stephen J. Evans ◽  
Nicole Hondow ◽  
Afshin Tarat ◽  
...  
Keyword(s):  
Dna Damage ◽  
Cultured Cells ◽  
Alveolar Epithelial Cell ◽  
Health And Safety ◽  
In Vitro Models ◽  
Type I ◽  
Toxicological Evaluation ◽  
Layer Graphene ◽  
Few Layer Graphene

Abstract Background Toxicological evaluation of engineered nanomaterials (ENMs) is essential for occupational health and safety, particularly where bulk manufactured ENMs such as few-layer graphene (FLG) are concerned. Additionally, there is a necessity to develop advanced in vitro models when testing ENMs to provide a physiologically relevant alternative to invasive animal experimentation. The aim of this study was to determine the genotoxicity of non-functionalised (neutral), amine- and carboxyl-functionalised FLG upon both human-transformed type-I (TT1) alveolar epithelial cell monocultures, as well as co-cultures of TT1 and differentiated THP-1 monocytes (d.THP-1 (macrophages)). Results In monocultures, TT1 and d.THP-1 macrophages showed a statistically significant (p < 0.05) cytotoxic response with each ENM following 24-h exposures. Monoculture genotoxicity measured by the in vitro cytokinesis blocked micronucleus (CBMN) assay revealed significant (p < 0.05) micronuclei induction at 8 µg/ml for amine- and carboxyl-FLG. Transmission electron microscopy (TEM) revealed ENMs were internalised by TT1 cells within membrane-bound vesicles. In the co-cultures, ENMs induced genotoxicity in the absence of cytotoxic effects. Co-cultures pre-exposed to 1.5 mM N-acetylcysteine (NAC), showed baseline levels of micronuclei induction, indicating that the genotoxicity observed was driven by oxidative stress. Conclusions Therefore, FLG genotoxicity when examined in monocultures, results in primary-indirect DNA damage; whereas co-cultured cells reveal secondary mechanisms of DNA damage.

scholarly journals Few-Layer Graphene Induces Both Primary and Secondary Genotoxicity in in Vitro Alveolar Barrier Models

2020 ◽  
Author(s):  
Michael John Burgum ◽  
Martin JD Cl ◽  
Stephen J Evans ◽  
Nicole Hondow ◽  
Afshin Tarat ◽  
...  
Keyword(s):  
Dna Damage ◽  
Cultured Cells ◽  
Alveolar Epithelial Cell ◽  
Health And Safety ◽  
Type I ◽  
Toxicological Evaluation ◽  
Alveolar Epithelial ◽  
Layer Graphene ◽  
Few Layer Graphene

Abstract Background: Toxicological evaluation of engineered nanomaterials (ENMs) is essential for occupational health and safety, particularly where bulk manufactured ENMs such as few-layer graphene (FLG) are concerned. Additionally, there is a necessity to develop advanced in vitro models when testing ENMs to provide a physiologically relevant alternative to invasive animal experimentation. The aim of this study was to determine the genotoxicity of non-functionalised (neutral), amine- and carboxyl-functionalised FLG upon both human-transformed type-I (TT1) alveolar epithelial cell monocultures, as well as co-cultures of TT1 and differentiated THP-1 monocytes (d.THP-1 (macrophages)). Results: In monocultures, TT1 and d.THP-1 macrophages showed a statistically significant (p<0.05) cytotoxic response with each ENM following 24-hour exposures. Monoculture genotoxicity measured by the in vitro cytokinesis blocked micronucleus (CBMN) assay revealed significant (p<0.05) micronuclei induction at 8µg/ml for amine- and carboxyl-FLG. Transmission electron microscopy (TEM) revealed ENMs were internalised by TT1 cells within membrane-bound vesicles. In the co-cultures, ENMs induced genotoxicity in the absence of cytotoxic effects. Co-cultures pre-exposed to 1.5mM N-acetylcysteine (NAC), showed baseline levels of micronuclei induction, indicating that the genotoxicity observed was driven by oxidative stress. Conclusions: Therefore, FLG genotoxicity when examined in monocultures, results in primary-indirect DNA damage; whereas co-cultured cells reveal secondary mechanisms of DNA damage.

Toxicological evaluation of MnAl based permanent magnets using different in vitro models

Chemosphere ◽  
2021 ◽  
Vol 263 ◽  
pp. 128343
Author(s):  
Carlos Rumbo ◽  
Cristina Cancho Espina ◽  
Vladimir V. Popov ◽  
Konstantin Skokov ◽  
Juan Antonio Tamayo-Ramos
Keyword(s):  
Permanent Magnets ◽  
In Vitro Models ◽  
Toxicological Evaluation

scholarly journals Role of collagenase in mediating in vitro alveolar epithelial wound repair

1999 ◽  
Vol 112 (2) ◽  
pp. 243-252
Author(s):  
E. Planus ◽  
S. Galiacy ◽  
M. Matthay ◽  
V. Laurent ◽  
J. Gavrilovic ◽  
...  
Keyword(s):  
Cell Migration ◽  
Cell Adhesion ◽  
Epithelial Cell ◽  
Type I Collagen ◽  
Alveolar Epithelial Cell ◽  
Cell Monolayer ◽  
Type I ◽  
Type Ii ◽  
Alveolar Epithelial

Type II pneumocytes are essential for repair of the injured alveolar epithelium. The effect of two MMP collagenases, MMP-1 and MMP-13 on alveolar epithelial repair was studied in vitro. The A549 alveolar epithelial cell line and primary rat alveolar epithelial cell cultures were used. Cell adhesion and cell migration were measured with and without exogenous MMP-1. Wound healing of a cell monolayer of rat alveolar epithelial cell after a mechanical injury was evaluated by time lapse video analysis. Cell adhesion on type I collagen, as well as cytoskeleton stiffness, was decreased in the presence of exogenous collagenases. A similar decrease was observed when cell adhesion was tested on collagen that was first incubated with MMP-1 (versus control on intact collagen). Cell migration on type I collagen was promoted by collagenases. Wound healing of an alveolar epithelial cell monolayer was enhanced in the presence of exogenous collagenases. Our results suggest that collagenases could modulate the repair process by decreasing cell adhesion and cell stiffness, and by increasing cell migration on type I collagen. Collagen degradation could modify cell adhesion sites and collagen degradation peptides could induce alveolar type II pneumocyte migration. New insights regarding alveolar epithelial cell migration are particularly relevant to investigate early events during alveolar epithelial repair following lung injury.

Mechanisms and regulation of ion transport in adult mammalian alveolar type II pneumocytes

1991 ◽  
Vol 261 (5) ◽  
pp. C727-C738 ◽  
Author(s):  
S. Matalon
Keyword(s):  
Epithelial Transport ◽  
Cultured Cells ◽  
Basolateral Membrane ◽  
Alveolar Epithelium ◽  
Alveolar Type ◽  
Type I ◽  
Type Ii ◽  
Epithelial Lining ◽  
Epithelial Lining Fluid

The adult alveolar epithelium consists of type I and type II (ATII) pneumocytes that form a tight barrier, which severely restricts the entry of lipid-insoluble molecules from the interstitial to the alveolar space. Current in vivo and in vitro evidence indicates that the alveolar epithelium is also an absorptive epithelium, capable of transporting Na+ from the alveolar lumen, which is bathed by a small amount of epithelial lining fluid, to the interstitial space. The in situ localization of Na(+)-K(+)-ATPase activity in ATII cells and the fact that these cells are involved in a number of crucial functions, such as surfactant secretion and alveolar remodeling after injury, led investigators to examine their transport characteristics. Radioactive flux studies, in both freshly isolated and cultured cells, and bioelectric measurements in ATII cells grown on porous supports indicate that they transport Na+ according to the Koefoed-Johnsen and Ussing model of epithelial transport. Na+ enters the apical membrane, because of the favorable electrochemical gradient, through Na+ cotransporters, a Na(+)-H+ antiport, and cation channels and is pumped across the basolateral membrane by a ouabain-sensitive Na(+)-K+ pump. Na+ transport is enhanced by substances that increase intracellular adenosine 3',5'-cyclic monophosphate. In addition to Na+ transporters, ATII cells contain several transporters that regulate their intracellular pH, including a H(+)-ATPase, which may explain the low pH of the epithelial lining fluid. The absorptive properties of ATII cells may play an important role in regulating the degree of alveolar fluid in health and disease.

scholarly journals Toxicological evaluation of highly water dispersible few-layer graphene in vivo

Carbon ◽  
2020 ◽  
Vol 170 ◽  
pp. 347-360
Author(s):  
Amalia Ruiz ◽  
Matteo Andrea Lucherelli ◽  
Diane Murera ◽  
Delphine Lamon ◽  
Cécilia Ménard-Moyon ◽  
...  
Keyword(s):  
Toxicological Evaluation ◽  
Water Dispersible ◽  
Layer Graphene ◽  
Few Layer Graphene

Injury of rat pulmonary alveolar epithelial cells by H2O2: dependence on phenotype and catalase

1991 ◽  
Vol 260 (4) ◽  
pp. L318-L325 ◽  
Author(s):  
R. H. Simon ◽  
J. A. Edwards ◽  
M. M. Reza ◽  
R. G. Kunkel
Keyword(s):  
Epithelial Cells ◽  
Catalase Activity ◽  
Lung Diseases ◽  
Alveolar Epithelial Cell ◽  
Alveolar Epithelial Cells ◽  
Type I ◽  
Type Ii Cells ◽  
Type Ii ◽  
Alveolar Epithelial

In a variety of inflammatory lung diseases, type I alveolar epithelial cells are more likely to be injured than are type II cells. Because oxidants have been implicated as a cause of injury in various inflammatory lung diseases, we evaluated the effects of differentiation on alveolar epithelial cell susceptibility to H2O2-induced injury. With the use of isolated rat type II cells in culture, we found that the cytotoxic effect of H2O2 increased between days 2 and 7, when type II cells are known to lose their distinctive type II properties and assume a more type I-like appearance. We previously reported that type II cells utilized both intracellular catalase and glutathione-dependent reactions to protect against H2O2. We therefore examined whether alterations in either of these protective mechanisms were responsible for the differentiation-dependent changes in sensitivity to H2O2. We found that catalase activity within alveolar epithelial cells decreased between 2 and 7 days in culture, whereas no changes were detected in glutathione-dependent systems. We then used a histochemical technique that detects catalase activity and found that type II cells within rat lungs possessed numerous catalase-containing peroxisomes, whereas very few were detected in type I cells. These findings demonstrate that as type II cells assume a type I-like phenotype, they become more susceptible to H2O2-induced injury. This increased susceptibility is associated with reductions in intracellular catalase activity, both in vitro and in vivo.

scholarly journals Modulation of T1α expression with alveolar epithelial cell phenotype in vitro

1998 ◽  
Vol 275 (1) ◽  
pp. L155-L164 ◽  
Author(s):  
Zea Borok ◽  
Spencer I. Danto ◽  
Richard L. Lubman ◽  
Yuxia Cao ◽  
Mary C. Williams ◽  
...  
Keyword(s):  
Epithelial Cell ◽  
Epithelial Cells ◽  
Cell Shape ◽  
Alveolar Epithelial Cell ◽  
Cell Phenotype ◽  
Alveolar Type ◽  
Type I ◽  
Soluble Factors ◽  
Alveolar Epithelial

T1α is a recently identified gene expressed in the adult rat lung by alveolar type I (AT1) epithelial cells but not by alveolar type II (AT2) epithelial cells. We evaluated the effects of modulating alveolar epithelial cell (AEC) phenotype in vitro on T1α expression using either soluble factors or changes in cell shape to influence phenotype. For studies on the effects of soluble factors on T1α expression, rat AT2 cells were grown on polycarbonate filters in serum-free medium (MDSF) or in MDSF supplemented with either bovine serum (BS, 10%), rat serum (RS, 5%), or keratinocyte growth factor (KGF, 10 ng/ml) from either day 0 or day 4 through day 8 in culture. For studies on the effects of cell shape on T1α expression, AT2 cells were plated on thick collagen gels in MDSF supplemented with BS. Gels were detached on either day 1(DG1) or day 4 (DG4) or were left attached until day 8. RNA and protein were harvested at intervals between days 1 and 8 in culture, and T1α expression was quantified by Northern and Western blotting, respectively. Expression of T1α progressively increases in AEC grown in MDSF ± BS between day 1 and day 8 in culture, consistent with transition toward an AT1 cell phenotype. Exposure to RS or KGF from day 0 prevents the increase in T1α expression on day 8, whereas addition of either factor from day 4 through day 8 reverses the increase. AEC cultured on attached gels express high levels of T1α on days 4 and 8. T1α expression is markedly inhibited in both DG1 and DG4 cultures, consistent with both inhibition and reversal of the transition toward the AT1 cell phenotype. These results demonstrate that both soluble factors and alterations in cell shape modulate T1α expression in parallel with AEC phenotype and provide further support for the concept that transdifferentiation between AT2 and AT1 cell phenotypes is at least partially reversible.

scholarly journals BH3-only proteins Puma and Bim are rate-limiting for γ-radiation– and glucocorticoid-induced apoptosis of lymphoid cells in vivo

Blood ◽  
2005 ◽  
Vol 106 (13) ◽  
pp. 4131-4138 ◽  
Author(s):  
Miriam Erlacher ◽  
Ewa M. Michalak ◽  
Priscilla N. Kelly ◽  
Verena Labi ◽  
Harald Niederegger ◽  
...  
Keyword(s):  
Dna Damage ◽  
Target Genes ◽  
Cultured Cells ◽  
Lymphoid Cells ◽  
Whole Body ◽  
Γ Radiation ◽  
B Cell Leukemia ◽  
Induced Apoptosis

Numerous p53 target genes have been implicated in DNA damage–induced apoptosis signaling, but proapoptotic Bcl-2 (B-cell leukemia 2) family members of the BH3 (Bcl-2 homolog region [BH] 3)–only subgroup appear to play the critical initiating role. In various types of cultured cells, 3 BH3-only proteins, namely Puma (p53 up-regulated modulator of apoptosis), Noxa, and Bim (Bcl-2 interacting mediator of cell death), have been shown to initiate p53-dependent as well as p53-independent apoptosis in response to DNA damage and treatment with anticancer drugs or glucocorticoids. In particular, the absence of Puma or Bim renders thymocytes and mature lymphocytes refractory to varying degrees to death induced in vitro by growth factor withdrawal, DNA damage, or glucocorticoids. To assess the in vivo relevance of these findings, we subjected mice lacking Puma, Noxa, or Bim to whole-body γ-radiation or the glucocorticoid dexamethasone and compared lymphocyte survival with that in wild-type and BCL2–transgenic mice. Absence of Puma or Bcl-2 overexpression efficiently protected diverse types of lymphocytes from the effects of γ-radiation in vivo, and loss of Bim provided lower but significant protection in most lymphocytes, whereas Noxa deficiency had no impact. Furthermore, both Puma and Bim were found to contribute significantly to glucocorticoid-induced killing. Our results thus establish that Puma and Bim are key initiators of γ-radiation– and glucocorticoid-induced apoptosis in lymphoid cells in vivo.

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