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Inflammatory and cytotoxic responses of an alveolar-capillary coculture model to silica nanoparticles: comparison with conventional monocultures.

Kasper J, Hermanns MI, Bantz C, Maskos M, Stauber R, Pohl C, Unger RE, Kirkpatrick JC - Part Fibre Toxicol (2011)

Bottom Line: Attention should primarily be focussed on SNP effects on biological barriers.This may mimic the early inflammatory events that take place in the pulmonary alveoli after aSNP inhalation.Furthermore, a number of apoptosis markers belonging to the intrinsic pathway were upregulated in the coculture following aSNP treatment.

View Article: PubMed Central - HTML - PubMed

Affiliation: University Medical Centre, Institute of Pathology, Mainz, Germany.

ABSTRACT

Background: To date silica nanoparticles (SNPs) play an important role in modern technology and nanomedicine. SNPs are present in various materials (tyres, electrical and thermal insulation material, photovoltaic facilities). They are also used in products that are directly exposed to humans such as cosmetics or toothpaste. For that reason it is of great concern to evaluate the possible hazards of these engineered particles for human health. Attention should primarily be focussed on SNP effects on biological barriers. Accidentally released SNP could, for example, encounter the alveolar-capillary barrier by inhalation. In this study we examined the inflammatory and cytotoxic responses of monodisperse amorphous silica nanoparticles (aSNPs) of 30 nm in size on an in vitro coculture model mimicking the alveolar-capillary barrier and compared these to conventional monocultures.

Methods: Thus, the epithelial cell line, H441, and the endothelial cell line, ISO-HAS-1, were used in monoculture and in coculture on opposite sides of a filter membrane. Cytotoxicity was evaluated by the MTS assay, detection of membrane integrity (LDH release), and TER (Transepithelial Electrical Resistance) measurement. Additionally, parameters of inflammation (sICAM-1, IL-6 and IL-8 release) and apoptosis markers were investigated.

Results: Regarding toxic effects (viability, membrane integrity, TER) the coculture model was less sensitive to apical aSNP exposure than the conventional monocultures of the appropriate cells. On the other hand, the in vitro coculture model responded with the release of inflammatory markers in a much more sensitive fashion than the conventional monoculture. At concentrations that were 10-100fold less than the toxic concentrations the apically exposed coculture showed a release of IL-6 and IL-8 to the basolateral side. This may mimic the early inflammatory events that take place in the pulmonary alveoli after aSNP inhalation. Furthermore, a number of apoptosis markers belonging to the intrinsic pathway were upregulated in the coculture following aSNP treatment. Analysis of the individual markers indicated that the cells suffered from DNA damage, hypoxia and ER-stress.

Conclusion: We present evidence that our in vitro coculture model of the alveolar-capillary barrier is clearly advantageous compared to conventional monocultures in evaluating the extent of damage caused by hazardous material encountering the principle biological barrier in the lower respiratory tract.

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Mitochondrial activity was measured via the MTS assay (A) and membrane integrity was determined by the LDH assay (B) for monocultures of H441 and ISO-HAS-1 on 96 well plates (conventional monoculture). Cells were incubated with aSNP (Ludox TM-40: light grey, NexSil20: dark grey) for 4 h in serum-free medium. aSNPs were then removed and cells were cultivated for further 20 h. The assays were conducted after both time points (4 h exposure and 4 h exposure with 20 h recovery). Data are depicted as percentage of the untreated control (A: MTS) or as percentage of the total LDH amount of the cells (B: LDH, lysis control). Results are shown as means ± S.D. (n = 6-9) of 2-3 independent experiments. *P < 0.05, **P < 0.01 and ***P < 0.001 compared to the untreated control
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Figure 1: Mitochondrial activity was measured via the MTS assay (A) and membrane integrity was determined by the LDH assay (B) for monocultures of H441 and ISO-HAS-1 on 96 well plates (conventional monoculture). Cells were incubated with aSNP (Ludox TM-40: light grey, NexSil20: dark grey) for 4 h in serum-free medium. aSNPs were then removed and cells were cultivated for further 20 h. The assays were conducted after both time points (4 h exposure and 4 h exposure with 20 h recovery). Data are depicted as percentage of the untreated control (A: MTS) or as percentage of the total LDH amount of the cells (B: LDH, lysis control). Results are shown as means ± S.D. (n = 6-9) of 2-3 independent experiments. *P < 0.05, **P < 0.01 and ***P < 0.001 compared to the untreated control

Mentions: Both particles NexSil20 and Ludox TM-40 at a concentration of 600 μg/ml (180 μg/cm2 cell layer) resulted in a cell viability of about 80% of the untreated control cells (Ludox TM-40: 85 ± 8.4% and NexSil20: 80 ± 7.9%) for the H441 cell line and about 40% (Ludox TM-40: 41 ± 6.5% and NexSil20: 37 ± 6%) for ISO-HAS-1 (MTS assay, Figure 1A) in the conventional monoculture after 4 h incubation. 300 μg/ml aSNP showed no significant effect on the viability of H441 (Ludox TM-40: 97 ± 8.4% and NexSil20: 95 ± 7%), whereas for the ISO-HAS-1 cells a reduced viability (Ludox TM-40: 32 ± 1.3% and NexSil20: 40 ± 3.5%) was still measurable. Thus, the endothelial cell line seems to be more sensitive to aSNP exposure than the epithelial cell line. After removal of the unattached aSNPs cell viability further decreased in a concentration-dependent fashion for both cell lines (see Figure 1A: 4 h exposure, 20 h recovery). A concentration of 60 μg/ml aSNPs or less did not show any effect on MTS conversion.


Inflammatory and cytotoxic responses of an alveolar-capillary coculture model to silica nanoparticles: comparison with conventional monocultures.

Kasper J, Hermanns MI, Bantz C, Maskos M, Stauber R, Pohl C, Unger RE, Kirkpatrick JC - Part Fibre Toxicol (2011)

Mitochondrial activity was measured via the MTS assay (A) and membrane integrity was determined by the LDH assay (B) for monocultures of H441 and ISO-HAS-1 on 96 well plates (conventional monoculture). Cells were incubated with aSNP (Ludox TM-40: light grey, NexSil20: dark grey) for 4 h in serum-free medium. aSNPs were then removed and cells were cultivated for further 20 h. The assays were conducted after both time points (4 h exposure and 4 h exposure with 20 h recovery). Data are depicted as percentage of the untreated control (A: MTS) or as percentage of the total LDH amount of the cells (B: LDH, lysis control). Results are shown as means ± S.D. (n = 6-9) of 2-3 independent experiments. *P < 0.05, **P < 0.01 and ***P < 0.001 compared to the untreated control
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3040689&req=5

Figure 1: Mitochondrial activity was measured via the MTS assay (A) and membrane integrity was determined by the LDH assay (B) for monocultures of H441 and ISO-HAS-1 on 96 well plates (conventional monoculture). Cells were incubated with aSNP (Ludox TM-40: light grey, NexSil20: dark grey) for 4 h in serum-free medium. aSNPs were then removed and cells were cultivated for further 20 h. The assays were conducted after both time points (4 h exposure and 4 h exposure with 20 h recovery). Data are depicted as percentage of the untreated control (A: MTS) or as percentage of the total LDH amount of the cells (B: LDH, lysis control). Results are shown as means ± S.D. (n = 6-9) of 2-3 independent experiments. *P < 0.05, **P < 0.01 and ***P < 0.001 compared to the untreated control
Mentions: Both particles NexSil20 and Ludox TM-40 at a concentration of 600 μg/ml (180 μg/cm2 cell layer) resulted in a cell viability of about 80% of the untreated control cells (Ludox TM-40: 85 ± 8.4% and NexSil20: 80 ± 7.9%) for the H441 cell line and about 40% (Ludox TM-40: 41 ± 6.5% and NexSil20: 37 ± 6%) for ISO-HAS-1 (MTS assay, Figure 1A) in the conventional monoculture after 4 h incubation. 300 μg/ml aSNP showed no significant effect on the viability of H441 (Ludox TM-40: 97 ± 8.4% and NexSil20: 95 ± 7%), whereas for the ISO-HAS-1 cells a reduced viability (Ludox TM-40: 32 ± 1.3% and NexSil20: 40 ± 3.5%) was still measurable. Thus, the endothelial cell line seems to be more sensitive to aSNP exposure than the epithelial cell line. After removal of the unattached aSNPs cell viability further decreased in a concentration-dependent fashion for both cell lines (see Figure 1A: 4 h exposure, 20 h recovery). A concentration of 60 μg/ml aSNPs or less did not show any effect on MTS conversion.

Bottom Line: Attention should primarily be focussed on SNP effects on biological barriers.This may mimic the early inflammatory events that take place in the pulmonary alveoli after aSNP inhalation.Furthermore, a number of apoptosis markers belonging to the intrinsic pathway were upregulated in the coculture following aSNP treatment.

View Article: PubMed Central - HTML - PubMed

Affiliation: University Medical Centre, Institute of Pathology, Mainz, Germany.

ABSTRACT

Background: To date silica nanoparticles (SNPs) play an important role in modern technology and nanomedicine. SNPs are present in various materials (tyres, electrical and thermal insulation material, photovoltaic facilities). They are also used in products that are directly exposed to humans such as cosmetics or toothpaste. For that reason it is of great concern to evaluate the possible hazards of these engineered particles for human health. Attention should primarily be focussed on SNP effects on biological barriers. Accidentally released SNP could, for example, encounter the alveolar-capillary barrier by inhalation. In this study we examined the inflammatory and cytotoxic responses of monodisperse amorphous silica nanoparticles (aSNPs) of 30 nm in size on an in vitro coculture model mimicking the alveolar-capillary barrier and compared these to conventional monocultures.

Methods: Thus, the epithelial cell line, H441, and the endothelial cell line, ISO-HAS-1, were used in monoculture and in coculture on opposite sides of a filter membrane. Cytotoxicity was evaluated by the MTS assay, detection of membrane integrity (LDH release), and TER (Transepithelial Electrical Resistance) measurement. Additionally, parameters of inflammation (sICAM-1, IL-6 and IL-8 release) and apoptosis markers were investigated.

Results: Regarding toxic effects (viability, membrane integrity, TER) the coculture model was less sensitive to apical aSNP exposure than the conventional monocultures of the appropriate cells. On the other hand, the in vitro coculture model responded with the release of inflammatory markers in a much more sensitive fashion than the conventional monoculture. At concentrations that were 10-100fold less than the toxic concentrations the apically exposed coculture showed a release of IL-6 and IL-8 to the basolateral side. This may mimic the early inflammatory events that take place in the pulmonary alveoli after aSNP inhalation. Furthermore, a number of apoptosis markers belonging to the intrinsic pathway were upregulated in the coculture following aSNP treatment. Analysis of the individual markers indicated that the cells suffered from DNA damage, hypoxia and ER-stress.

Conclusion: We present evidence that our in vitro coculture model of the alveolar-capillary barrier is clearly advantageous compared to conventional monocultures in evaluating the extent of damage caused by hazardous material encountering the principle biological barrier in the lower respiratory tract.

Show MeSH
Related in: MedlinePlus